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PubMed2002

Asymmetric synthesis of (-)-swainsonine, (+)-1,2-di-epi-swainsonine, and (+)-1,2,8-tri-epi-swainsonine.

The asymmetric synthesis of (-)-swainsonine via a nonchiral pool route that involves the Sharpless epoxidation to induce chirality is reported. The key steps involve vinyl epoxide aminolysis, ring-closing metathesis, and intramolecular N-alkylation to prepare the indolizidine ring and a highly diastereoselective cis-dihydroxylation using AD-mix-alpha. This synthetic strategy also allowed for the diastereoselective synthesis of (+)-1,2-di-epi-swainsonine and (+)-1,2,8-tri-epi-swainsonine.

Alkylation↗

PubMed2002

Appearance and disappearance of swainsonine in serum and milk of lactating ruminants with nursing young following a single dose exposure to swainsonine (locoweed; Oxytropis sericea).

A series of experiments were conducted to investigate the elimination of swainsonine in the milk of lactating ruminants following a single dose oral exposure to swainsonine (locoweed; Oxytropis sericea) and to assess subsequent subclinical effects on the mothers and their nursing young. In a preliminary experiment, lactating ewes were gavaged with locoweed providing 0.8 mg swainsonine/kg BW (n = 4; BW = 75.8 +/- 3.6 kg; lactation = d 45) and lactating cows were offered up to 2.0 mg swainsonine/kg BW free choice (n = 16; BW = 389.6 +/- 20.9 kg; lactation = d 90). Serum and milk were collected at h 0 (before treatment), 3, 6, 12, and 24 for ewes, and h 0 (before treatment), 6, 12, 18, and 24 for cows. Swainsonine was highest (P < 0.05) by h 6 in the serum and milk of ewes. Consumption of at least 0.61 mg swainsonine/kg BW induced consistent (> 0.025 microg/mL) appearance of swainsonine in cow serum and milk. In response to the results obtained in the preliminary experiment, a subsequent experiment utilizing lactating ewes (n = 13; BW = 74.8 +/- 6.4 kg; lactation = d 30) and cows (n = 13; BW = 460.8 +/- 51.9 kg; lactation = d 90) was conducted. Each lactating ruminant was gavaged with a locoweed extract to provide 0 (control), 0.2, or 0.8 mg swainsonine/kg BW and individually penned with her nursing young. Serum and milk from the mothers and serum from the nursing young were collected at h 0 (before treatment), 3, 6, 9, 12, 24 and 48 (an additional sample was obtained at h 72 for ewes and lambs). Serum and milk swainsonine was higher (P < 0.05) in the 0.8 mg treated groups and maximal (P < 0.05) concentrations occurred from h 3 to 6 for ewes and h 6 to 12 h for cows (P < 0.05). Rises in alkaline phosphatase activity indicated subclinical toxicity in the treated ewes (P < 0.05). Following a single dose oral exposure to 0.2 and 0.8 mg swainsonine/kg BW provided by a locoweed extract, swainsonine was detected in the serum and milk of lactating ewes and cows, and rises in serum alkaline phosphatase activity were observed in the ewes. Neither swainsonine nor changes in alkaline phosphatase activity was detected in the serum of the lambs and calves nursing the ewes and cows dosed with swainsonine.

Alkaline Phosphatase↗

PubMed1989

Structure-activity relationship of swainsonine. Inhibition of human alpha-mannosidases by swainsonine analogues.

The inhibitory properties of a series of synthetic epimers and analogues of swainsonine towards the multiple forms of human alpha-mannosidases were studied in vitro and in cells in culture. Of the five epimers tested, only the 8a-epimer and 8,8a-diepimer of swainsonine were specific and competitive inhibitors (Ki values of 7.5 x 10(-5) and 2 x 10(-6) M respectively) of lysosomal alpha-mannosidases in vitro and induced storage of mannose-rich oligosaccharides in human fibroblasts in culture. The structures of these storage products indicated that processing alpha-mannosidases had also been inhibited. This was consistent with the observed inhibition in vitro of these enzymes by these compounds. In contrast, the 8-epimer, 1,8-diepimer and 2,8a-diepimer of swainsonine had no appreciable effect on any alpha-mannosidases. The corresponding open-chain analogues of swainsonine, namely 1,4-dideoxy-1,4-imino-D-mannitol, of the 8a-epimer, namely 1,4-dideoxy-1,4-imino-D-talitol, and of the 8,8a-diepimer, namely 1,4-dideoxy-1,4-imino-L-allitol, were weaker competitive inhibitors of lysosomal alpha-mannosidase, with Ki values of 1.3 x 10(-5), 1.2 x 10(-4) and 1.2 x 10(-4) M respectively. These analogues also proved less effective at inducing oligosaccharide accumulation and in disturbing glycoprotein processing. These compounds offer the opportunity to determine which alterations in the chirality of the swainsonine molecule affect its inhibitory specificity. A comparison of their biological activities has identified reagents that will be useful for studying steps in the biosynthesis and catabolism of glycoproteins and that may be of potential value in chemotherapy.

Alkaloids↗

PubMed1985

Studies of an immunomodulator, swainsonine. II. Effect of swainsonine on mouse immunodeficient system and experimental murine tumor.

We examined the effect of swainsonine on antibody response to sheep red blood cells (SRBC) in immunodeficient mice which were treated with immunosuppressive factor or antitumor drugs, or inoculated with sarcoma 180 ascites tumor. The administration of swainsonine restored the capacities of the immunodeficient mice to produce antibody against SRBC. Furthermore, swainsonine inhibited completely the growth of sarcoma 180 ascites tumor in mice. Swainsonine also reduced lung metastases of B16 melanoma in mice. These results suggest that swainsonine has potential as an immunomodulator for the treatment of immunocompromised hosts.

Adjuvants, Immunologic↗

PubMed2000

Asymmetric syntheses of (-)-8-epi-swainsonine triacetate and (+)-1, 2-Di-epi-swainsonine. Carbonyl addition thwarted by an unprecedented aza-pinacol rearrangement.

Indolizidines (-)-8-epi-swainsonine triacetate and (+)-1, 2-di-epi-swainsonine were synthesized from the O'Donnell Schiff base ester 1 derived from D-serine. Reductive-alkenylation of 1 with (i)()Bu(5)Al(2)H/H(2)C=CHMgBr followed by substrate-directed dihydroxylation of the pendant allylic group with OsO(4), reduction of imine, and cyclization with Ph(3)P/CCl(4) gave the polyhydroxylated pyrrolidines 8a and 8b as advanced intermediates. Efficient protecting group manipulations converted pyrrolidines 8a and 8b to their corresponding partially protected analogues 10a and 10b, which upon Swern oxidation and diastereoselective Keck-type allylation with BF(3).Et(2)O afforded the required three-carbon homologues (10a, >20:1 de; 10b, 3.5:1 de). Use of the chelating Lewis acid MgBr(2) instead of BF(3).Et(2)O with 10a led to a novel aza-pinacol rearrangement and allylation at the alpha-carbon to yield amino alcohol 17, which is similar to a hydride migration in the biosynthetic pathway of indolizidine alkaloids. Subsequent hydroboration, cyclization, and deprotection furnished (-)-8-epi-swainsonine triacetate 15a and (+)-1,2-di-epi-swainsonine 16b in good overall yields (6.3% for 1 --> 15a, 13 steps, and 4.0% for 1 --> 16b, 14 steps).

Alkylation↗

PubMed1985

Studies of an immunomodulator, swainsonine. I. Enhancement of immune response by swainsonine in vitro.

Swainsonine isolated from Metarhizium sp., was found to enhance the activities of the mouse immune system in vitro. Concanavalin A stimulated lymphocyte proliferation and proliferative response in mixed lymphocytes culture, which were suppressed by immunosuppressive factor obtained from serum of sarcoma 180 tumor bearing mouse, were restored to normal levels by the addition of swainsonine. Furthermore, the concanavalin A induced incorporation of [3H]thymidine into mouse spleen cells was remarkably increased by treatment with swainsonine over a wide dose range, From studies using fluorescence activated cell sorting, swainsonine was shown to enhance the expression of concanavalin A receptors of spleen cells.

Adjuvants, Immunologic↗

PubMed1983

The uptake of swainsonine, a specific inhibitor of alpha-D-mannosidase, into normal human fibroblasts in culture.

Swainsonine, an indolizidine alkaloid, found in plants of the genus Swainsona, has been shown to be a strong inhibitor in vitro of the alpha-D-mannosidase activity in normal human fibroblasts. Therefore, inhibition of alpha-D-mannosidase activity in extracts of harvested cells grown with swainsonine in the medium has been used to follow the association of the alkaloid with normal human fibroblasts in culture. Swainsonine that could not be removed by extensive washing became associated with the cells within 1 min, and it is concluded that the alkaloid is internalized rapidly by the cells. The amount of swainsonine taken up into the cells depended on the length of time in contact and the concentration of swainsonine in the medium, but at 37 degrees C a plateau of internalized swainsonine occurred after 2 hr with extracellular concentrations of swainsonine of 100 microM or greater. At lower concentrations of swainsonine the rate of uptake was found to be temperature-dependent, increasing greatly at 20 degrees C. The rapidity and temperature sensitivity of the uptake, together with the observation that mannose or mannose-6-phosphate did not prevent the association, suggest that swainsonine enters the cells by permeation rather than by endocytosis. When swainsonine is withdrawn from the culture medium, there is a decrease with time of cell-associated swainsonine. The kinetics of uptake and release of swainsonine and its slightly basic nature make it likely that swainsonine is concentrated initially in the lysosomes. This rapid, but reversible, concentration of swainsonine in lysosomes would be consistent with the observed effects of the toxin in vivo.

Alkaloids↗

PubMed1995

Tissue and serum swainsonine concentrations in sheep ingesting Astragalus lentiginosus (locoweed).

Locoweed intoxication or locoism results when animals continuously graze certain plants of the general Astragalus or Oxytropis. The locoweed toxin, swainsonine, is water soluble and is rapidly absorbed and eliminated. The purpose of this study was to determine the distribution of swainsonine in tissues of sheep eating locoweed and to determine if the tissue swainsonine concentrations change with continued locoweed ingestion. Fifteen cross-breed whethers were divided into 3 groups of 5 each and fed alfalfa pellets (Group 1) or alfalfa pellets with 10% Astragalus lentiginosus for 13 d (Group 2) or for 21 d (Group 3). After the feeding periods, the animals were slaughtered and tissues were collected, frozen and later analyzed for swainsonine using an in vitro, alpha-mannosidase inhibition assay. Significant alpha-mannosidase inhibitory activity (interpreted as ng/ml of swainsonine) was detected in whole blood, skeletal muscle, brain, kidney, liver, thyroid and urine. The swainsonine concentrations in tissues were significantly correlated with daily swainsonine intake (r = 0.58 to 0.96). With the exception of kidney, longer exposure did not result in significant increases in the swainsonine concentrations in blood, muscle, brain, liver or thyroid. Liver had the highest swainsonine concentrations with 3049 +/- 1952 and 3947 +/- 457 ng/ml (mean +/- SD) in Groups 2 and 3 respectively. Swainsonine concentrations varied widely within the groups suggesting individual animal variability in swainsonine absorption, metabolism and excretion. These findings suggest that swainsonine is present in tissues of animals eating locoweed and that in most tissues the amount was directly correlated to the swainsonine dose ingested, but not to the length of exposure.

Animals↗

PubMed2006

Inhibition of the growth of human gastric carcinoma in vivo and in vitro by swainsonine.

In Europe, swainsonine has been studied widely for prevention of metastasis and cancer therapy. In order to investigate the effects and mechanisms of swainsonine on the human gastric carcinoma SGC-7901 cell, we carried out in vivo and in vitro experiments. After treatment with swainsonine, an effective dose and IC50 value of swainsonine for SGC-7901 cells were examined by MTT assay. Cell-cycle distribution and apoptotic rates were analyzed using FCM, and [Ca2+]i was measured using LSCM. The expression of p53, c-myc and Bcl-2 were determined using an immunocytochemical method. Simultaneously, 50 mice were divided randomly into five groups. Three groups were administrated swainsonine at dose of 3, 6 and 12 mg/kg body wt., two control groups were administrated N.S. 20 ml/kg body wt. and 5-Fu 20 mg/kg body wt., respectively, by intraperitoneal injection. The inhibition rate was calculated and pathological sections were observed. The growth of SGC-7901 cell is inhibited by swainsonine in vitro, with an IC50 value at 24 h of 0.84 microg/ml, and complete inhibition concentration is 6.2 microg/ml. After treatment with swainsonine at the concentrations of 0.5, 1.5 and 4.5 microg/ml for 24 h, the expression of apoptosis inhibiting gene p53 and bcl-2 decreases, and the apoptotic trigger gene c-myc increases markedly (p<0.05), as well as [Ca2+]i overloading, SGC-7901 cell is induced to apoptosis in the end. It is also found that the percentages of S phase are 38.8%, 39.7% and 29.6%, respectively (20.0% in control group and 23.2% in 5-Fu group). The rates of inhibition were 13.2%, 28.9%, 27.3%, respectively, when the nude mice were administered swainsonine (p<0.05 or 0.01). The structure of the tumor showed hemorrhage, necrosis and inflammatory cell infiltration. We therefore conclude that swainsonine could inhibit cell proliferation in vitro and the growth of human gastric carcinoma in vivo. The mechanisms of swainsonine-induced apoptosis may relate to [Ca2+]i overloading and the expression of apoptosis-related genes.

Animals↗

PubMed1987

Effect of isomers of swainsonine on glycosidase activity and glycoprotein processing.

The chemical synthesis of swainsonine [(1S,2R,8R,8 alpha R)-trihydroxyindolizidine] from trans-1,4-dichloro-2-butene was previously described [Adams, C. E., Walker, F. J., & Sharpless, K. B. (1985) J. Org. Chem. 50, 420-424]. A modification of that synthesis provided two other isomers, referred to here as "Glc-swainsonine" [(1S,2S,8R,8 alpha R)-trihydroxyindolizidine] and "Ido-swainsonine" [(1S,2S,8S,8 alpha R)-trihydroxyindolizidine]. To determine whether these new compounds had biological activity, they were compared to swainsonine as inhibitors of a number of commercially available glycosidases. While swainsonine is a potent inhibitor of jack bean alpha-mannosidase but does not inhibit other glycosidases, its two isomers were inactive on alpha-mannosidase but did inhibit other enzymes. Thus, Glc-swainsonine was an inhibitor of the fungal alpha-glucosidase amyloglucosidase, and this inhibition was of a competitive nature (Ki = 5 X 10(-5) M) with respect to the substrate p-nitrophenyl alpha-D-glucopyranoside. This alkaloid also inhibited beta-glucosidase, but much less effectively than alpha-glucosidase. On the other hand, Ido-swainsonine was more effective toward beta-glucosidase than toward alpha-glucosidase, and this inhibition was also of a competitive nature. None of these inhibitors were effective against beta-mannosidase or alpha- or beta-galactosidase. Glc-swainsonine was also tested against the glycoprotein processing glycosidases. Surprisingly, in this respect, the alkaloid was like swainsonine in that it inhibited mannosidase II but had no effect or only slight effect on glucosidase I, glucosidase II, and mannosidase I. Glc-swainsonine also inhibited glycoprotein processing in cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1998

In vitro effect of swainsonine on bovine and ovine lymphoblastogenesis.

Peripheral blood lymphocytes (PBL) were obtained from heifers and wethers to assess the in vitro effects of swainsonine. Stimulated and unstimulated PBL (2x10(5)/well; 96 well plates) were incubated in the presence of swainsonine (2, .2, .02, .002, and 0 microg/mL; n = 32) for 72 h. Mitogens used to stimulate PBL were concanavalin A (ConA; 5 microg/mL), phytohemagglutinin-P (PHA-P; 5 microg/mL), phytohemagglutinin-M (PHA-M; 5 microg/mL), pokeweed mitogen (PWM; 5 microg/mL), and lipopolysaccharide (LPS; 10 microg/mL). Controls received no swainsonine or mitogen. Effects of swainsonine are expressed as a percentage of stimulation relative to control (stimulation index; SI). Unstimulated bovine PBL treated with 2 microg/mL of swainsonine exhibited a depressed (P<.001; SI of 87 and 100 for 2 microg/mL and control, respectively; SE = 2.7) SI. At concentrations of .2 and 2 microg/mL, swainsonine inhibited (P<.001; SI of 351, 310, and 464 for .2, 2, and 0 microg/mL, respectively; SE = 27.1) bovine PBL proliferative response to PHA-P. Swainsonine had a mitogenic effect on unstimulated ovine PBL at .2 and 2 microg/mL (P<.05; SI of 118, 113, and 100 for .2 and 2 microg/mL and control, respectively; SE = 4.4). Swainsonine inhibited ovine proliferative responses to PHA-P at .2 and 2 microg/mL (P<.005; SI of 190, 178, and 228 for .2, 2, and 0 microg/mL, respectively; SE = 9.4) and to PHA-M at .002 and 2 microg/mL (P<.03; SI of 165, 167, and 192 for .002, 2, and 0 microg/mL, respectively; SE = 7.9). The opposing responses of unstimulated ovine (mitogenic) and bovine (antiproliferative) PBL to swainsonine indicates a differential species response. Swainsonine suppression of PHA-P-induced proliferation would indicate a negative effect on ovine and bovine T-cell function.

Adjuvants, Immunologic↗

PubMed1998

Tissue swainsonine clearance in sheep chronically poisoned with locoweed (Oxytropis sericea).

Locoweed poisoning is seen throughout the world and annually costs the livestock industry millions of dollars. Swainsonine inhibits lysosomal alpha-mannosidase and Golgi mannosidase II. Poisoned animals are lethargic, anorexic, emaciated, and have neurologic signs that range from subtle apprehension to seizures. Swainsonine is water-soluble, rapidly absorbed, and likely to be widely distributed in the tissues of poisoned animals. The purpose of this study was to quantify swainsonine in tissues of locoweed-poisoned sheep and determine the rate of swainsonine clearance from animal tissues. Twenty-four crossbred wethers were gavaged with ground Oxytropis sericea to obtain swainsonine doses of 1 mg swainsonine x kg(-1) BW x d(-1) for 30 d. After dosing, the sheep were killed on d 0, 1, 2, 3, 4, 6, 14, 30, 60, and 160. Animal weights and feed consumption were monitored. Serum was collected during dosing and withdrawal periods, and tissues were collected at necropsy. Serum swainsonine concentrations were determined using an alpha-mannosidase inhibition assay. Swainsonine concentrations in skeletal muscle, heart, brain, and serum were similar at approximately 250 ng/g. Clearance from these tissues was also similar, with half-lives (T(1/2)) of less than 20 h. Swainsonine at more than 2,000 ng/g, was detected in the liver, spleen, kidney, and pancreas. Clearance from liver, kidney, and pancreas was about T(1/2) 60 h. These findings imply that poisoned sheep have significant tissue swainsonine concentrations and animals exposed to locoweed should be withheld from slaughter for at least 25 d (10 T(1/2)) to ensure that the locoweed toxin has cleared from animal tissues and products.

Animals↗

PubMed1986

Effects of swainsonine and polyinosinic:polycytidylic acid on murine tumor cell growth and metastasis.

Increased sialylation and branching of asparagine-linked oligosaccharides have recently been associated with both neoplastic transformation and the metastatic phenotype. Swainsonine, an inhibitor of Golgi alpha-mannosidase II blocks the synthesis of sialylated tri- and tetraantennary asparagine-linked oligosaccharides and results in the expression of hybrid-type oligosaccharides at the cell surface. Both the lymphoid tumor line MDAY-D2 and B16F10 melanoma cells were less metastatic when grown in swainsonine (0.3 micrograms/ml) for 48 h prior to injection of the cells into the lateral tail veins of mice. The addition of swainsonine (2.5 micrograms/ml) to the drinking water of the mice further reduced the incidence of lung colonization by B16F10 melanoma cells. MDAY-D2 tumors removed from mice on swainsonine-supplemented drinking water showed a loss of leukoagglutinin-binding complex-type oligosaccharides similar to that of tumor cells cultured in medium containing swainsonine. The growth rate of s.c. MDAY-D2 tumors was not reduced by the addition of swainsonine to the drinking water of the host; however, when mice were given two i.p. injections of the interferon-inducing agent polyinosinic:polycytidylic acid in addition to swainsonine, the primary tumor grew at a reduced rate compared to either treatment alone. Swainsonine alone did not inhibit tumor cell growth in vitro; however, the drug enhanced the antiproliferative effect of interferon. The survival time of mice bearing established MDAY-D2 metastases was extended by treating the animals with swainsonine and polyinosinic:polycytidylic acid; however, the number of long-term survival was unchanged. Swainsonine-treated tumor cells appeared to be compromised in two ways: reduced organ colonization potential; and drug-treated MDAY-D2 cells were more sensitive to the antiproliferative effects of interferon in vitro and in vivo.

Alkaloids↗

PubMed1984

Swainsonine and castanospermine blockade of mannose glycoprotein uptake by macrophages. Apparent inhibition of receptor-mediated endocytosis by endogenous ligands.

Receptor-mediated uptake of mannose-terminated glycoproteins by macrophages is blocked by treating the cells with swainsonine, an inhibitor of alpha-mannosidase II, and by castanospermine, an inhibitor of the endoplasmic reticulum processing enzyme alpha-glucosidase. Both inhibitors are known to cause accumulation of unprocessed oligosaccharide chains terminating in mannose. Inhibition of ligand uptake by the drugs was time- and dose-dependent. Swainsonine produced a maximal effect after 2 h; castanospermine required 5-6 h. Following swainsonine treatment, complete recovery of mannose receptor activity required 24 h and was blocked by cycloheximide suggesting that new receptor synthesis was necessary. Tunicamycin, an inhibitor of oligosaccharide assembly, had no effect on uptake of mannosylated ligands, but tunicamycin pretreatment reduced the sensitivity to swainsonine. These effects of swainsonine and castanospermine appear to be specific, other macrophage pinocytosis receptors (e.g. mannose phosphate) or phagocytosis of yeast particles were unaffected. Moreover, swainsonine had no effect on the fibroblast mannose phosphate receptor. The ability of macrophages to process newly synthesized oligosaccharides was blocked following treatment with swainsonine. Normal processing was fully recovered 24 h after removal of the drug. Mannosidase II was partially inactivated by swainsonine treatment and only a portion was recovered after 24 h. Treatment of macrophages with swainsonine also resulted in an increase in net lysosomal enzyme secretion. Inhibition of mannose-specific receptor-mediated endocytosis in macrophages by swainsonine and castanospermine appears to be due to the formation of mannose-terminated membrane glycoproteins which engage the mannose receptor thereby preventing function. These results suggest a novel mechanism for regulation of receptor-mediated endocytosis.

Alkaloids↗

PubMed1994

A phase I study of swainsonine in patients with advanced malignancies.

Swainsonine, an alpha-mannosidase inhibitor which blocks Golgi oligosaccharide processing, represents a new class of compounds that inhibit both rate of tumor growth, and metastasis, in murine experimental tumor models. In this first phase I study, the quantitative and qualitative toxicities of swainsonine have been studied in patients given a continuous i.v. infusion over 5 days, repeated at 28-day intervals. Dose levels were escalated in increments of 100 micrograms/kg/day from 50-550 micrograms/kg/day. Nineteen patients with both solid tumor and hematological malignancies were given a total of 31 courses. Hepatotoxicity, particularly in patients with liver metastases, was the dose-limiting toxicity. The maximum tolerated dose (MTD) and the recommended starting dose (MTD -1 level) were 550 and 450 micrograms/kg/day, respectively. Common side effects included edema, mild liver dysfunction, a rise in serum amylase, and decreased serum retinol. Acute respiratory distress syndrome possibly precipitated by swainsonine resulted in a treatment-related death in a patient with significant pretreatment hepatic dysfunction. One patient with head and neck cancer showed > 50% shrinkage of tumor mass for 6 weeks after treatment. Two patients with lymphangitis carcinomatosis on chest X-ray noted improvement in cough and shortness of breath during the infusion of swainsonine and for 1 week thereafter. Clearance and serum half-life for swainsonine were determined to be approximately 2 ml/h/kg, and 0.5 day, respectively. Golgi oligosaccharide processing, a putative anticancer target for swainsonine was inhibited in peripheral blood lymphocytes as evidenced by a marked decrease in leukoagglutinin binding after 5 days of treatment. Oligomannosides in patient urine increased 5-to 10-fold over the 5 days of treatment, indicating that tissue lysosomal alpha-mannosidases were also blocked by swainsonine. Urine oligomannoside accumulation reached steady state at 3 days, approximately 1 day after serum drug levels reached steady state. The fraction of HLA-DR-positive cells in peripheral blood lymphocytes increased following 5 days of swainsonine treatment, an effect similar to that observed for peripheral blood lymphocytes from normal subjects cultured with swainsonine. No significant changes in CD3, CD4, CD8, CD16, and CD25 were observed. Swainsonine produces minimal toxicity when administered i.v. to cancer patients at dosages that inhibit both Golgi alpha-mannosidase II and lysosomal alpha-mannosidases. Detection of hepatic metastases or liver enzyme abnormalities prior to treatment predict for more significant toxicity.

Adult↗

PubMed2001

Analysis of swainsonine: extraction methods, detection, and measurement in populations of locoweeds (Oxytropis spp.).

An analytical method has been developed to measure the locoweed toxin, swainsonine, in locoweed plant material. Dry ground plant samples were extracted using a small-scale liquid/liquid extraction procedure followed by isolation of the swainsonine by solid phase extraction with a cation-exchange resin. Detection and quantitation of the swainsonine were accomplished using reversed phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (LC-MS(2)). The limit of quantitation was estimated to be 0.001% swainsonine by weight in dry plant material, which corresponds to the lower threshold for toxicity of locoweeds. The method of analysis was applied to the analysis of Oxytropis sericea (white locoweed) and Oxytropis lambertii (Lambert locoweed) plant samples to measure the variability of individual plant swainsonine levels within populations and within species. Individual plant variability was found to be highly significant for both O. sericea and O. lambertii populations. The combined three-year mean swainsonine values taken from three populations of O. sericea ranged from 0.046% in Utah to 0.097% in a New Mexico population. Sixteen individual populations of O. lambertii were sampled from eight different U.S. states. Swainsonine was detected at levels >0.001% in only 5 of the 16 collection sites. Those populations of O. lambertii found to contain higher swainsonine levels were restricted to the most southern and western portion of its distribution, and all were identified as belonging to var. bigelovii, whereas var. articulata and var. lambertii samples contained swainsonine at levels <0.001%.

Chromatography, High Pressure Liquid↗

PubMed1997

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae.

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 microg ml(-1). Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.

2-Aminoadipic Acid↗

PubMed1999

Swainsonine protects both murine and human haematopoietic systems from chemotherapeutic toxicity.

The haematopoietic system is sensitive to cytotoxic damage and is often the site of dose-limiting toxicity. We previously reported that swainsonine, an inhibitor of protein glycosylation, reduced the bone marrow toxicity resulting from a single dose of anticancer drugs in otherwise healthy mice. However, more important questions are (1) can swainsonine protect tumour-bearing mice without interfering with the anti-tumour effects of the drugs, and (2) can swainsonine stimulate haematopoietic activity of human, as well as murine, bone marrow. We demonstrate here that swainsonine protects C57BL/6 mice bearing melanoma-derived tumours from cyclophosphamide-induced toxicity without interfering with the drug's ability to inhibit tumour growth. Similar results were obtained in vivo with 3'-azido-3'-deoxythymidine (AZT), a myelosuppressive agent often used in therapy for acquired immune deficiency syndrome. Swainsonine increased both total bone marrow cellularity and the number of circulating white blood cells in mice treated with doses of AZT that typically lead to severe myelosuppression. Swainsonine also increased the number of erythroid and myeloid colony forming cells (CFCs) in short-term cultures of murine bone marrow, restoring the number of progenitor cells to the control level in the presence of AZT doses that reduced CFCs by 80%. With respect to the sensitivity of human haematopoietic cells to swainsonine, we show that swainsonine protected human myeloid progenitor cells from AZT toxicity in vitro. These results suggest that swainsonine may be useful as an adjuvant in several types of human chemotherapy.

Adjuvants, Immunologic↗