Pyrrolizidine Alkaloids-Pros and Cons for Pharmaceutical and Medical Applications.

Heterocyclic organic compounds named pyrrolizidine alkaloids (PAs) belong to a group of alkaloids and are synthesized by either plants or microorganisms. Therefore, they are naturally occurring secondary metabolites. They are found in species applied in the pharmaceutical and food industries, thus a thorough knowledge of their pharmacological properties and toxicology to humans is of great importance for their further safe employment. This review is original because it synthesizes knowledge of plant and microbial PAs, which is unusual in the scientific literature. We have focused on the Boraginaceae family, which is unique due to the exceptional richness and diversity of its PAs in plant species. We have also presented the microbial sources of PAs, both from fungi and bacteria. The structure and metabolism of PAs have been discussed. Our main aim was to summarize the effects of PAs on humans, including both negative, toxic ones, mainly concerning hepatotoxicity and carcinogenicity, as well as potentially positive ones for pharmacological and medical applications. We have collected the results of studies on the anticancer activity of PAs from plant and microbial sources (mainly Streptomyces strains) and on the antimicrobial activity of PAs on different strains of microorganisms (bacteria and fungi). Finally, we have suggested potential applications and future perspectives.

A novel tricyclic BTK inhibitor suppresses B cell responses and osteoclastic bone erosion in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by joint leukocyte infiltration, synovial inflammation and bone damage result from osteoclastogenesis. Bruton's tyrosine kinase (BTK) is a key regulator of B cell receptor (BCR) and Fc gamma receptor (FcγR) signaling involved in the pathobiology of RA and other autoimmune disorders. SOMCL-17-016 is a potent and selective tricyclic BTK inhibitor, structurally distinct from other known BTK inhibitors. In present study we investigated the therapeutic efficacy of SOMCL-17-016 in a mouse collagen-induced arthritis (CIA) model and underlying mechanisms. CIA mice were administered SOMCL-17-016 (6.25, 12.5, 25 mg·kg-1·d-1, ig), or ibrutinib (25 mg·kg-1·d-1, ig) or acalabrutinib (25 mg·kg-1·d-1, ig) for 15 days. We showed that oral administration of SOMCL-17-016 dose-dependently ameliorated arthritis severity and bone damage in CIA mice; it displayed a higher in vivo efficacy than ibrutinib and acalabrutinib at the corresponding dosage. We found that SOMCL-17-016 administration dose-dependently inhibited anti-IgM-induced proliferation and activation of B cells from CIA mice, and significantly decreased anti-IgM/anti-CD40-stimulated RANKL expression in memory B cells from RA patients. In RANKL/M-CSF-stimulated RAW264.7 cells, SOMCL-17-016 prevented osteoclast differentiation and abolished RANK-BTK-PLCγ2-NFATc1 signaling. In summary, this study demonstrates that SOMCL-17-016 presents distinguished therapeutic effects in the CIA model. SOMCL-17-016 exerts a dual inhibition of B cell function and osteoclastogenesis, suggesting that it to be a promising drug candidate for RA treatment.

Lycopsamine inhibits the proliferation of human lung cancer cells via induction of apoptosis and autophagy and suppression of interleukin-2.

PURPOSE: Lycopsamine is an active pyrrolizidine alkaloid that shows significant bioactivity. Herein, lycopsamine was evaluated for the first time for its anti-lung cancer activity. Its effects on cellular apoptosis, autophagy, cell cycle and IL-2 gene were also examined. METHODS: The human lung cancer A549 and normal MRC5 cells were used in the study. MTT assay was used to determine the cytotoxicity of lycopsamine. Transmission electron microscopy (TEM) and western blotting were implemented for analyzing autophagy. DAPI staining, Annexin V/FITC/Propidium iodide (PI) and western blotting assays were used to study cellular apoptosis. Cell cycle was examined through flow cytometry. The expression of IL-2 gene was monitored by western blotting. RESULTS: Lycopsamine targeted the proliferation rate and reduced it remarkably in a dose-dependent manner. On searching for underlying mechanism, the antiproliferative effect of lycopsamine was due to autophagy and the expressions of pro-autophagy proteins (LC3-I, LC3-II, Beclin-1) increased on drug exposure. Furthermore, the antiproliferative effects were also found to be mediated via apoptosis induction and were associated with increased Bax and decreased Bcl-2 levels. Next, flow cytometry showed that lycopsamine inhibited cell cycle progression at G2/M-check point in lung cancer cells. Furthermore, the expressions of IL-2 gene decreased after lycopsamine treatment of these cells. In conclusion, on testifying the current designed hypothesis, lycopsamine showed significant antiproliferative effects in A549 lung cancer cells in a dose reliant manner. The antiproliferative effects of lycopsamine were associated with its autophagy inducing, apoptosis inducing, and inhibiting IL-2 expression, potential. CONCLUSION: Taken together, lycopsamine is a potent anti-lung cancer agent and can be a lead molecule in lung cancer treatment.

Radioiodination and in vivo assessment of the potential of newly synthesized pyrrolizine-5-carboxamides derivative in tumor model.

Recently, pyrrolizine derivatives have been reported to possess numerous anticancer activities. In a previous study, (EZ)-6-((4-chlorobenzylidene)-amino)-7-cyano-N-(p-tolyl)-2,3-dihydro-1H-pyrrolizine carboxamide (EZPCA) compound was synthesized and the cytotoxic activity of EZPCA toward COX-2 enzyme (overexpressed in cancer cells) was reported. In order to assess the suitability of this compound as a promising pilot structure for in vivo applications, EZPCA was radiolabeled with radioiodine-131 (131I) and various factors affecting radiolabeling process were studied. Quality control studies of [131I]iodo-EZPCA were performed using paper chromatography and HPLC was used as a co-chromatographic technique for confirming the radiochemical yield. Biodistribution studies of [131I]iodo-EZPCA were undertaken in normal and tumor bearing mice. The radiochemical yield percentage of [131I]iodo-EZPCA was 94.20 ± 0.12%. The biodistribution results showed evident tumor uptake of [131I]iodo-EZPCA with promising target/non-target (T/NT) ratios. As a conclusion, these data suggest that [131I]iodo-EZPCA had high binding efficiency, high tumor uptake and sufficient stability to be used be used in diagnostic studies.

Successful Use of Entecavir in Hepatitis B-associated Membranous Nephropathy.

We report the case of a 7-year-old unimmunized boy who presented with generalized anasarca for the first time, along with nephrotic-range proteinuria, hypoalbuminemia, microscopic hematuria and hypertension. Special investigations revealed ELISA test to be positive for hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg); hepatitis B viral DNA load (HBV DNA) level (real-time polymerase chain reaction) was 54 360 903 IU/ml. For hepatitis B virus (HBV)-related glomerulopathy, he was started on enalapril and lasilactone, and percutaneous renal biopsy was performed, which revealed membranous nephropathy (MN). A diagnosis of MN secondary to HBV infection contracted via horizontal transmission was made. The patient was started on peginterferon alfa-2b (50 µg/week) for 24 weeks. He failed to attain remission and seroconversion after interferon (IFN) therapy. Then, oral therapy with entecavir was started, and he attained remission as well as seroconversion after 3 months of therapy. He maintained his seroconversion status at his 6-month and the recent 12-month (quantitative HBV DNA level was 373 IU/ml) follow-up visit. Entecavir seems a promising drug for HBV-related glomerulopathy, especially in IFN-resistant cases.

Toxic phytochemicals and their potential risks for human cancer.

Consuming plants for their presumed health benefits has occurred since early civilizations. Phytochemicals are found in various plants that are frequently included in the human diet and are generally thought to be safe for consumption because they are produced naturally. However, this is not always the case and in fact many natural compounds found in several commonly consumed plants are potential carcinogens or tumor promoters and should be avoided.

Insight into HIV of IFN-induced myxovirus resistance 2 (MX2) expressed by traditional Chinese medicine.

Recently, an important topic of the acquired immunodeficiency syndrome (AIDS) had been published in 2013. In this report, the expression of the IFN-induced myxovirus resistance 2 (MX2) had been defined the function to kill the human immunodeficiency virus (HIV). The screening from the Traditional Chinese Medicine (TCM) database by simulating molecular docking and molecular dynamics could select candidate compounds, which may express MX2 against HIV. Saussureamine C, Crotalaburnine, and Precatorine are selected based on the highest docking score and other TCM compounds. The data from molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions, and hydrogen bond with structure variations, this research could assess the interaction between protein and ligand interaction. In addition to the detection of TCM compound efficacy, we suggest that Saussureamine C is better than the others in protein-ligand interaction and the structural variation to express MX2.

Plants containing pyrrolizidine alkaloids used in the traditional Indian medicine--including ayurveda.

Pyrrolizidine alkaloids (PAs) show a hazardous potential for humans and animals. They can possess mutagenic, teratogenic, cancerogenic and fetotoxic properties. One pathway of a human intoxication can be the use of medicinal plants which contain toxic PAs. The Traditional Indian medicine--in particular Ayurveda--is a popular and well-known healing system. Within this system several PA-containing plants are used which, on account of their PA level, represent a severe health risk. In general, it is not recommended to use plants containing those toxic compounds.

Repopulation by endogenous hepatocytes does not reconstitute liver mass in rats treated with retrorsine.

The retrorsine (RS)-based model for massive liver repopulation was laid on the hypothesis that transplanted cells can proliferate in the recipient liver if the growth capacity of endogenous hepatocytes is persistently impaired. In order to directly test this hypothesis, we examined the long-term response to 2/3 partial hepatectomy (PH) in rats pretreated with RS, according to the protocol for liver repopulation. Rats were given RS or saline and 4 weeks later they underwent PH; they were killed up to 16 weeks thereafter. Liver weights, liver DNA, and protein content were significantly lower in the RS group throughout the experimental time considered (e.g., at 16 weeks post-PH relative liver weight was 1.99 +/- 0.30% in RS group vs. 3.06 +/- 0.5% in controls). Regenerative nodules were present in RS-treated livers; they occupied about 3% of the liver at 2 weeks post-PH and this value increased to nearly 50% at 8 weeks and to > 95% at 16 weeks. In conclusion, RS-treated rat liver is unable to recover its original mass for several months following PH, despite the development of regenerative nodules. This long-lasting effect is likely to contribute to the growth of transplanted hepatocytes, leading to massive liver repopulation.

Preventive activity of pyrrolizidine alkaloids from Seneciobrasiliensis (Asteraceae) on gastric and duodenal induced ulcer on mice and rats.

The alkaloid extract of Senecio brasiliensis inflorescences contain a mixture of the pyrrolizidine alkaloids (PA) senecionine, integerrimine, retrorsine, usaramine and seneciphylline. We evaluated this PA mixture on preventive antiulcerogenic effects on standard rodent models of induced gastric and duodenal ulcers. In the HCl/ethanol, indomethacin-bethanechol and hypothermic-restraint-induced gastric ulcer, the lesion was significantly inhibited by PA (p.o.) (p < 0.001). In the pylorus-ligature, PA (i.d.), significantly increased the gastric juice content and the pH values and decreased the acid output. In the cysteamine induced duodenal ulcers, PA (p.o.) showed significant inhibition (p < 0.001) of the duodenal lesions when compared to the respective control. The levels of the somatostatin hormone in the blood samples of animals pre-treated with the PA (12.5 mg/kg) and the free mucus and prostaglandin synthesis also increased (p < 0.001) after administration of PA extract (p.o.). The results suggested that the PA extract from Senecio brasiliensis inflorescences presents a significant anti-ulcer effect in the selected ulcer models. The mechanism involved with the action of the PA extract is the cytoprotection. Additional studies are in progress to determine other possible mechanisms involved with effect of the PA as anti-ulcer agents.

Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkaloid, and metabolites in rats and mice.

Riddelliine is a representative pyrrolizidine alkaloid, a class of naturally occurring toxic phytochemicals present in plant species worldwide. Human exposure to pyrrolizidine alkaloids occurs through consumption of herbal dietary supplements, including comfrey, and through contaminated livestock products (e.g., milk). A recently completed 2-year bioassay of riddelliine carcinogenicity showed that male and female rats and male mice, but not female mice, developed liver tumors. The toxicokinetics of riddelliine and two metabolites, the N-oxide and retronecine, were determined in serum following an oral gavage dose in male and female rats and mice using a validated liquid chromatography-electrospray mass spectrometric method. The results are consistent with extensive metabolism of riddelliine and its more polar metabolites prior to excretion. It is concluded that factors other than toxicokinetics are responsible for the observed species/sex specificity of gross toxicity or liver tumor induction in rats and mice.

New cytotoxic agents, BE-54238A and B, produced by a streptomycete.

New cytotoxic substances, designated BE-54238A and B, were isolated from the culture broth of Streptomyces sp. A54238. The active principles were extracted from the mycelium by methanol and purified by Diaion HP-20 and Sephadex LH-20 column chromatographies. BE-54238A and B exhibited cytotoxic activity against murine and human tumor cell lines.

Hepatotoxic pyrrolizidine alkaloids in the Mexican medicinal plant Packera candidissima (Asteraceae: Senecioneae).

A study of the hepatotoxic pyrrolizidine alkaloids (PAs) contained in Packera candidissima (Greene) Weber & Löve (Senecio candidissimus Greene), a Mexican medicinal plant used for the treatment of kidney ailments and noted for its antiseptic properties, is reported. Analysis by TLC and GC-MS have shown the presence in high levels of both PAs and their N-oxides in the root (0.76% dry weight) and the aerial parts (0.36% dry weight) of the plant material. GC-MS analysis showed the presence of senecionine, integerrimine, retrorsine, and usaramine in the aerial parts. The root was found to contain senkirkine as the major component. These results indicate that users of this herb are at high risk of poisoning, especially members of certain cultural groups in Northern Mexico and the Hispanic population in the southwestern United States. In addition, two sesquiterpenes, neoadenostylone and epineoadenostylone were identified from the neutral extracts of this plant material. The 6 alpha-angeloyloxy-9-oxo-delta 10(1)-furanoeremophilane is reported for the first time as a natural product.

Phase II trial of indicine N-oxide in relapsed acute leukemia of childhood. A report from the Childrens Cancer Study Group.

We treated 31 children with acute lymphoblastic leukemia (ALL), 14 children with acute nonlymphoblastic leukemia (ANLL) in relapse, and 1 child with chronic myelogenous leukemia (CML) in blast crisis (CALLA negative) with indicine N-oxide in a Phase II study. The efficacy and toxicity of the drug were assessed at two dose levels: 2,000 mg/m2/day for 5 consecutive days (14 patients) and 2,500 mg/m2/day for 5 consecutive days (17 patients). One patient with ALL at each dose level achieved a complete response (CR) lasting 6 months and 1 month, respectively. The patient with CML achieved a partial response lasting 4 months. None of the patients with ANLL achieved a CR. Hepatotoxicity was mild (grade 1 or 2) in 63% and moderate (grade 3) in 9% of mild (grade 1 or 2) in 63% and moderate (grade 3) in 9% of patients; 3 patients (9%) experienced severe hepatotoxicity. Although indicine N-oxide has some antileukemic activity in ALL and is safe at the doses used in this study, the antileukemic activity is significantly less at these two doses than at greater than or equal to 3,000 mg/m2/days for 5 consecutive days. Unfortunately, when the higher doses are administered to children, they are associated with an unacceptably high incidence of severe, irreversible hepatotoxicity.

Phase II trial of indicine N-oxide in relapsed pediatric solid tumors. A report from the Childrens Cancer Study Group.

We used indicine N-oxide to treat 46 children with malignant solid tumors: 17 with osteosarcoma, 12 with neuroblastoma, 13 with a brain tumor, and 4 with other miscellaneous tumors. The efficacy and toxicity of the drug was assessed at the dose of 2000 mg/m2/day for five consecutive days. None of the 39 patients evaluable for response achieved a complete or partial response. Hepatotoxicity was experienced by 13 patients: 11 patients developed asymptomatic elevations of transaminases, 1 patient developed hyperbilirubinemia, and 1 developed ascites. Indicine N-oxide appears to be ineffective in the treatment of osteosarcoma, neuroblastoma, and pediatric brain tumors at this dose and schedule. Because higher doses are associated with an unacceptably high incidence of severe, irreversible hepatotoxicity, we do not recommend further study of this agent in pediatric solid tumors.

Changes in contractile and noncontractile protein metabolisms in both ventricles in monocrotaline-treated rats.

To clarify the metabolism of contractile and noncontractile proteins of both ventricles (BVs) during the development of right ventricular hypertrophy (RVH) induced by pressure overload, monocrotaline (M) was injected subcutaneously into Sprague-Dawley (SD) rats. Myosin isoenzymes (MIEs) were analyzed by pyrophosphate gel electrophoresis. Acid-soluble collagens were analyzed using improved noninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tissue collagen concentrations were also measured. M induced RVH, but not left ventricular hypertrophy, at 2 weeks, and severe RVH at 4 weeks. In right ventricles (RVs) of M-treated rats, MIE significantly shifted from V1 to V3, and the proportions of type III and V collagens increased compared to control at 2 and 4 weeks. In the left ventricles (LVs) of M-treated rats, similar but less remarkable MIE shifts were found without remodeling of collagen types at 2 and 4 weeks. Collagen concentrations of BVs treated with M did not show any significant changes compared to control at 2 and 4 weeks. Our results show remodelings of contractile and noncontractile proteins in RVs during the development of RVH, and also provide evidence for the changes in protein metabolism of the counterpart of RVs (i.e., LVs) during the development of cardiac hypertrophy.

Phase I trial of indicine-N-oxide in children with leukemia and solid tumors: a Pediatric Oncology Group study.

A phase I trial of indicine-N-oxide was carried out in 12 children with solid tumors and in 16 with leukemia. Doses of 5, 6, and 7.5 g/m2 were given parenterally as a 15-min infusion every 3 weeks. The maximum tolerated dose in patients with solid tumors was 7.5 g/m2 and the dose-limiting toxicity was myelosuppression. In leukemia, the maximum tolerated dose was 6.0 g/m2 and hepatotoxicity was dose-limiting. Half of the children with leukemia showed elevations in transaminase levels and one child died of massive hepatic necrosis. This hepatotoxicity limits the use of indicine-N-oxide in children with leukemia. Antineoplastic activity was limited to a transient reduction in the numbers of circulating leukemic cells.

Semisynthetic pyrrolizidine alkaloid N-oxide antitumor agents. Esters of heliotridine.

The C-9 and C-7 monoesters and C-7, C-9 diesters of heliotridine with (S)-(+) and (R)-(-)-2-hydroxy-2-phenylbutyric acid were prepared, converted into their N-oxides, and compared with the corresponding C-9 monoesters of retronecine in the in vivo P388 lymphocytic leukemia screen. Relative in vitro cytotoxicities of some of the free bases and their corresponding N-oxides were also measured against the A204 rhabdomyosarcoma cell line by using the soft agar colony forming assay. Stereochemistry at C-7 of the necine and at C-2' of the necine acid appears to have a significant effect on the antitumor activity in this system. In the heliotridine series, the configuration of the necic acid has a pronounced effect on the site selectivity (C-7 vs C-9) in esterification with carbodiimidazole. An explanation for this site selectivity is offered.

Hepatocellular toxicity during the treatment of refractory acute leukemia with indicine N-oxide.

Indicine N-oxide is the first member of the large class of compounds comprised of pyrrolizidine alkaloids and their N-oxides to be studied in the treatment of cancer in humans. Twenty-two patients with refractory acute leukemia received indicine N-oxide daily for 5 consecutive days in a dose-seeking study. Of eight patients with refractory acute lymphocytic leukemia, one had a complete remission, and one had a partial remission. Of 11 patients with refractory acute nonlymphocytic leukemia, 2 patients had complete remissions. Of three patients with blast crisis of chronic granulocytic leukemia, one patient had a partial remission. Five patients had severe hepatic toxicity, probably due to veno-occlusive disease induced by the drug. Whether hepatotoxicity and antileukemic activity are a result of the same mechanism of action of indicine N-oxide is not known.