Cancer Lett. 2005 Jan 31;218(1):21-31.

Numerous anti-nflammatory agents have been shown to exert chemopreventive activity by targeting cyclooxygenase (COX)-2, a rate-limiting enzyme involved in the inflammatory process. Sutherlandia frutescens (L.) R. Br. and Harpagophytum procumbens DC., which are commonly known as Cancer bush (CB) and Devil's claw (DC), respectively, have long been used in South Africa for the management of pain and inflammation. In the present study, we investigated the effects of methanolic extracts of CB and DC on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in mouse skin. Topical application of both extracts inhibited TPA-induced COX-2 expression. As an underlying mechanism of COX-2 inhibition, these extracts diminished TPA-stimulated catalytic activity of extracellular signal-regulated protein kinase (ERK), which is known to regulate the activation of eukaryotic transcription factors mediating COX-2 induction. While TPA-induced activation of nuclear factor-kappaB remained unaffected by both extracts, they inhibited TPA-induced activation of activator protein-1 (AP-1) and attenuated the expression of its key component c-Fos. In another study, topical application of TPA induced DNA binding of cyclic AMP response element binding (CREB) protein in mouse skin in vivo, which was abrogated by pretreatment with either CB or DC.

Biofactors. 2004;21(1-4):149-53.

J Ethnopharmacol. 2005 Jan 4;96(1-2):113-9.

A screening process was applied to extracts made from Sutherlandia frutescens (L.) R. Br (Fabaceae) and Lobostemon trigonus (Boraginaceae) as identified by the Botany Department, University of Port Elizabeth to detect if any of the extracts inhibited the human immunodeficiency virus (HIV). For purposes of dereplication, sulphated polysaccharides were removed and bovine serum albumin (BSA) was included in the assays to adsorb non-specific tannins potentially present. In the reverse transcriptase (RT) assay, an aqueous extract of the Lobostemon leaves inhibited HIV-1 RT with an IC50 value of 49 microg/ml, while in the protease assay no inhibition was seen. In the alpha- and beta-glucosidase assays, no significant inhibition was seen with the inclusion of BSA, indicating tannin-based inhibitory effects on these two enzymes. The beta-glucuronidase inhibitory activity, however, was retained in the presence of BSA. The study shows that Sutherlandia extracts contain inhibitory compounds active against HIV target enzymes , while aqueous Lobostemon leaf extracts contain a potent HIV-1 RT inhibitor, thus showing a potential mechanistic action of these plants in aiding HIV-positive patients.

J Ethnopharmacol. 2004 Nov;95(1):1-5.

One of the best-known multi-purpose medicinal plants in Southern Africa, Sutherlandia frutescens subsp. microphylla (family: Fabaceae/Leguminosa), is used for a wide range of conditions, including cancer, viral diseases and inflammatory conditions. Little scientific data has been documented on the mechanism by which Sutherlandia frutescens acts on the immune system. Phagocyte derived reactive oxygen species, such as hydrogen peroxide and superoxide radicals, are responsible for the pathogenesis of various inflammatory conditions. Anti-inflammatory properties of various medicinal-plant extracts have been explained, at least in part, by their antioxidant activities. We investigated the effects of a hot water extract of Sutherlandia frutescens on both luminol and lucigenin enhanced chemiluminescence of neutrophils stimulated with L-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) as well as its superoxide and hydrogen peroxide scavenging properties in a cell free system. The results indicate that Sutherlandia frutescens extract possesses superoxide as well as hydrogen peroxide scavenging activities at concentrations as low as 10 microg/ml, which could account for some of the anti-inflammatory properties that have been described.

Methods Find Exp Clin Pharmacol. 2004 Jul-Aug;26(6):409-16.

Previous studies on the pharmacology of South African medicinal plants in our laboratories and elsewhere have shown that some plants possess therapeutic attributes. One such ethnomedically useful plant is Sutherlandia frutescens R. BR. (family: Fabaceae). S. frutescens is widely used in South African traditional medicine for the management and/or control of a plethora of human ailments. In order to scientifically appraise some of the ethnomedical uses of S. frutescens, the present study was undertaken to investigate the analgesic, antiinflammatory and antidiabetic properties of the plant's shoot aqueous extract in experimental animal models. The analgesic effect of the herb's shoot extract was evaluated using the hot-plate and acetic acid test models of pain in mice, while the antiinflammatory and hypoglycemic effects of the plant's shoot aqueous extract were investigated in rats, using fresh egg albumin-induced pedal (paw) edema, and streptozotocin (STZ)-induced diabetes mellitus. Diclofenac (100 mg/kg) and chlorpropamide (250 mg/kg) were used, respectively, as reference drugs for comparison. S. frutescens shoot aqueous extract (50-800 mg/kg i.p.) produced significant (p < 0.05-0.001) analgesic effects against thermally- and chemically-induced nociceptive pain stimuli in mice. The plant extract (50-800 mg/kg p.o. or i.p.) also significantly (p < 0.05-0.001) inhibited induced acute inflammation and caused significant (p < 0.05-0.001) hypoglycemia in rats. The various chemical constituents and secondary metabolites of the herb are speculated to account for the observed analgesic, antiinflammatory and hypoglycemic effects of the plant. The results of this experimental animal study suggest that S. frutescens shoot aqueous extract possesses analgesic, antiinflammatory, and hypoglycemic properties, and thus lend pharmacological credence to the suggested folkloric uses of the herb in the management and/or control of painful, arthritic and other inflammatory conditions, as well as for adult-onset, type-2 diabetes mellitus in some communities of South Africa.

J Ethnopharmacol. 2004 Jul;93(1):9-19.

In vitro culture studies of Sutherlandia frutescens on human tumor cell lines.

Tai J, Cheung S, Chan E. Departments of Pathology and Pediatrics, Center for Complementary Medicine Research, BC's Research Institute for Children's and Women's Health, University of British Columbia, Vancouver, BC, Canada.

Sutherlandia frutescens is a South African herb used traditionally by the natives to treat cancer, and more recently to improve the overall health in HIV/AIDS patients. Gas chromatography/mass spectrometer profiling and liquid chromatographic/mass spectral investigation confirmed and quantified the presence of canavanine, GABA and arginine in the herbal preparation used in this study. In vitro study demonstrated a concentration dependent effect of Sutherlandia on several tumor cell lines, with 50% inhibition (IC50) of proliferation of MCF7, MDA-MB-468, Jurkat and HL60 cells at 1/250, 1/200, 1/150 and 1/200 dilutions, respectively. Sutherlandia treatment did not induce HL60 differentiation along the macrophage/monocyte or granulocyte lineage. It demonstrated antioxidant activity in reducing free radical cations with an estimated activity of 0.5 microl of Sutherlandia extract equivalent to that of 10 microM of Trolox. However, it did not significantly suppress lipopolysaccharide stimulated nitric oxide production by murine macrophage/monocyte RAW 264.7 cells, nor did it significantly inhibit IL-1beta and TNF-alpha mRNA expression in RAW 264.7 cells. In conclusion, Sutherlandia ethanolic extract showed a concentration dependent antiproliferative effect on several human tumor cell lines but did not show significant antioxidant effects. Further studies are needed to explore the activities of this multipurpose South African herbal preparation.

Eur J Clin Nutr. 2004 Nov 10

Effects of pinitol isolated from soybeans on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus: a randomized controlled study.

Kim JI, Kim JC, Kang MJ. 1Biohealth Product Research Center, School of Food and Life Science, Institute for Food Sciences, Institute of Basic Sciences, Inje University, Gimhae, Republic of Korea.

OBJECTIVE:: To assess the effects of soybean-derived pinitol on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus. DESIGN:: Randomized, double-blind, placebo-controlled, parallel-group trial. SETTING:: Pusan Paik Hospital, Pusan, Republic of Korea. INTERVENTIONS:: A total of 30 patients with type II diabetes received an oral dose of 600 mg soybean-derived pinitol or placebo twice daily for 13 weeks. RESULTS:: Pinitol significantly decreased mean fasting plasma glucose, insulin, fructosamine, HbA(1c,) and the homeostatic model assessment insulin resistance index (HOMA-IR, P<0.001). Pinitol significantly decreased total cholesterol, LDL-cholesterol, the LDL/HDL-cholesterol ratio, and systolic and diastolic blood pressure and increased HDL-cholesterol (P<0.05). CONCLUSIONS:: These data suggest that soybean-derived pinitol may be beneficial in reducing cardiovascular risk in Korean type II diabetes.

Fitoterapia. 2001 Feb;72(2):168-70.

Anti-inflammatory effect of (+)-pinitol.

Singh RK, Pandey BL. Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

In the carrageenin-induced paw oedema in rats, (+)-pinitol (2.5-10 mg/kg, i.p.), isolated from Abies pindrow leaves, showed a significant anti-inflammatory effect, the highest dose being comparable to phenylbutazone (100 mg/kg, i.p.).

Br J Pharmacol. 2000 Aug;130(8):1944-8.

Insulin-like effect of pinitol.

Bates SH, Jones RB, Bailey CJ. School of Life and Health Sciences, Aston University, Birmingham, USA.

D-pinitol (3-O-methyl-chiroinositol), an active principle of the traditional antidiabetic plant Bougainvillea spectabilis, is claimed to exert insulin-like effects. This study investigates the effect of D-pinitol on glucose homeostasis in animal models of diabetes, and on glucose transport by cultured muscle cells. Plasma glucose concentrations were measured in normal, obese-diabetic (ob/ob) and streptozotocin (STZ)-diabetic mice after oral (p.o.) and intraperitoneal (i.p.) administration of D-pinitol. Glucose transport was measured in L6 rat muscle cells by 2-deoxyglucose (2DG) uptake. In STZ-diabetic mice, 100 mg kg(-1) p.o. D-pinitol acutely decreased the hyperglycaemia (by 22% at 6 h). A similar decrease in plasma glucose (by 21%) was observed after 100 mg kg(-1) i.p. D-pinitol. Insulin concentrations and the rate of insulin-induced (1 unit kg(-1) actrapid i.p.) glucose disappearance were not altered by 100 mg kg(-1) p.o. D-pinitol. Chronic administration of D-pinitol (100 mg kg(-1) i.p. twice daily for 11 days) to STZ-diabetic mice maintained a reduction in plasma glucose concentrations from about 14 to 10 mmol l(-1). In normal non-diabetic and severely insulin resistant ob/ob mice, 100 mg kg(-1) p.o. D-pinitol did not significantly affect plasma glucose or insulin during acute studies. Incubation of L6 muscle cells with D-pinitol (10(-3) M) increased basal 2DG uptake by 41% after 10 min and by 34% after 4 h. The effect of D-pinitol was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. D-pinitol did not increase insulin-stimulated 2DG uptake by L6 cells. The data support the view that D-pinitol can exert an insulin-like effect to improve glycaemic control in hypoinsulinaemic STZ-diabetic mice. D-pinitol may act via a post-receptor pathway of insulin action affecting glucose uptake.

Br Poult Sci. 2003 Sep;44(4):620-5.

L-canavanine inhibits L-arginine uptake by broiler chicken intestinal brush border membrane vesicles.

Rueda E, Michelangeli C, Gonzalez-Mujica F. Centro de Bioquimica Nutricional, Facultad de Ciencias Veterinarias, Universidad Central de Venezuela, Maracay, Estado Aragua, Venezuela.

1. Intestinal brush border membrane vesicles (BBMV) were prepared from 3-week-old broiler chickens. 2. Electron microscopy of the BBMV fraction showed single membrane vesicles of different sizes with no electron dense material inside. No other organelles were observed. The sucrase and maltase activities were enriched by factors of 16 and 18, respectively, in the BBMV fraction in comparison with the homogenate. On the other hand, the Na+/K+-ATPase sensitivity to ouabain was increased by a factor of 0.8. 3. The BBMV showed a maximum L-[14C]-arginine uptake (944.9 +/- 22.9 pmoles/mg protein) at 45 s and thereafter it declined slowly. In the presence of 0.5 mM L-canavanine, the L-[14C]-arginine uptake by BBMV was reduced by 43.6% at 45 s. 4. It is concluded that L-canavanine inhibits L-arginine Na+-dependent transport across the enterocyte apical membrane in a highly purified intestinal BBMV from broiler chickens.

Mol Cell Biochem. 2003 Feb;244(1-2):37-43.

L-Canavanine as a radiosensitization agent for human pancreatic cancer cells.

Bence AK, Adams VR, Crooks PA. Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA.

This study evaluated the in vitro effect of L-canavanine on cell cycle progression in the two human pancreatic cancer cells lines PANC-1 and MIA PaCa-2. After 72 h of exposure to L-canavanine, the percentage of cells in the radiosensitive G2/M phase of the cell cycle increased 6-fold in PANC-1 cells and 4-fold in MIA PaCa-2 cells, when compared to untreated cells. The capacity of L-canavanine to redistribute cells into the G2/M phase of the cell cycle was both concentration- and time-dependent. Since many drugs that cause cells to accumulate in the G2/M phase of the cell cycle are effective radiosensitization agents, the potential of L-canavanine to synergistically enhance the effects of ionizing radiation also was evaluated. The interaction between these treatment modalities was quantified using the median-effect equation and combination index analysis. L-Canavanine was found to be synergistic with radiation when either PANC-1 or MIA PaCa-2 cells were exposed to L-canavanine for 72 h prior to irradiation. These results suggest that L-canavanine in combination with radiation may have clinical potential in the treatment of pancreatic cancer.

Biochem Biophys Res Commun. 2002 Jul 12;295(2):283-8.

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL.

Jang MH, Jun do Y, Rue SW. Laboratory of Immunobiology, Department of Microbiology, College of Natural Sciences, Kyungpook National University, Taegu, Republic of Korea.

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.

Anticancer Drugs. 1999 Jan;10(1):113-8.

Growth inhibitory effect of L-canavanine against MIA PaCa-2 pancreatic cancer cells is not due to conversion to its toxic metabolite canaline.

Swaffar DS, Ang CY. Division of Basic Pharmaceutical Sciences, School of Pharmacy, Northeast Louisiana University, Monroe, USA.

L-Canavanine (CAV) is an arginine (ARG) analog isolated from the jack bean, Canavalia ensiformis. CAV becomes incorporated into cellular proteins of MIA PaCa-2 human pancreatic cancer cells and the aberrant, canavanyl proteins are not preferentially degraded. Hydrolytic cleavage of CAV to canaline (CAN) and urea is mediated by arginase. CAN is a potent metabolite that inactivates vitamin B6-containing enzymes and may inhibit cell growth. To determine the presence of arginase and its specificity for ARG and CAV in MIA PaCa-2 cells, a radiometric assay, which quantifies the 14C released from the hydrolytic cleavage of L-[guanidino-14C]ARG or L-[guanidinooxy-14C]CAV mediated by arginase, was employed. Insignificant amounts of 14CO2 were released when cells were exposed to [14C]CAV or to [14C]ARG. Pancreatic cancer cells secrete a negligible amount of arginase. Cytotoxic effects of CAN and CAV were compared on cells exposed to varying concentrations of ARG. These studies provide evidence that CAV's cytotoxic effects on MIA PaCa-2 cells cannot be attributed to conversion to the active metabolite CAN. A slower and decreased hydrolysis of CAV by arginase allows CAV to persist and increases its chances of incorporating into proteins in these cells. Lack of considerable amounts of arginase in the pancreas makes CAV a worthy candidate for further studies in pancreatic cancer.

Anticancer Drugs. 2002 Mar;13(3):313-20.

The antiproliferative and immunotoxic effects of L-canavanine and L-canaline.

Bence AK, Worthen DR. Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, USA.

L-Canavanine and its arginase-catalyzed metabolite, L-canaline, are two novel anticancer agents in development. Since the immunotoxic evaluation of agents in development is a critical component of the drug development process, the antiproliferative effects of L-canavanine and L-canaline were evaluated in vitro. Both L-canavanine and L-canaline were cytotoxic to peripheral blood mononucleocytes (PBMCs) in culture. Additionally, the mononucleocytes were concurrently exposed to either L-canavanine or L-canaline and each one of a series of compounds that may act as metabolic inhibitors of the action of L-canavanine and L-canaline (L-arginine, L-ornithine, D-arginine, L-lysine, L-homoarginine, putrescine, L-omega-nitro arginine methyl ester and L-citrulline). The capacity of these compounds to overcome the cytotoxic effects of L-canavanine or L-canaline was assessed in order to provide insight into the biochemical mechanisms that may underlie the toxicity of these two novel anticancer agents. The results of these studies suggest that the mechanism of L-canavanine toxicity is mediated through L-arginine-utilizing mechanisms and that the L-canavanine metabolite, L-canaline, is toxic to human PBMCs by disrupting polyamine biosynthesis. The elucidation of the biochemical mechanisms associated with the effects of L-canavanine and L-canaline on lymphoproliferation may be useful for maximizing the therapeutic effectiveness and minimizing the toxicity of these novel anticancer agents.

J Nippon Med Sch. 2002 Feb;69(1):13-8.

Effect of L-canavanine, an Inhibitor of inducible nitric oxide synthase, on myocardial dysfunction during septic shock.

Suzuki N, Sakamoto A. Department of Anesthesiology, Nippon Medical School, Tokyo, Japan.

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) plays a role in the pathophysiology of septic shock. The depression of cardiac contractility in such situations is mediated by proinflammatory cytokines, including interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). The effects of two NOS inhibitors with different isoform selectivity were compared in isolated working rat hearts. The depression of contractility by IL-1beta and TNF-alpha was prevented by administration of a nonselective nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) or an inhibitor of inducible nitric oxide synthase, L-canavanine. In contrast, when L-NAME was administered in the absence of IL-1beta and TNF-alpha, it depressed contractility over the 2h perfusion period by significantly reducing coronary flow. These results support current thinking that the depression of myocardial function by IL-1beta and TNF-alpha is mediated, at least in part, by an intracardiac increase in inducible nitric oxide synthase, and that in contrast to L-NAME, the decline in coronary conductance seen in cytokine-treated is not prevented by L-canavanine hearts. L-canavanine shows selective inhibition of inducible nitric oxide synthase unlike the vasopressor action of L-NAME in cytokine-treated hearts.

Cancer Lett. 1998 Oct 23;132(1-2):229-39.

L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines.

Worthen DR. Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, USA.

L-Canavanine (L-CAV) is a naturally occurring L-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 microM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 microM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.

Jpn J Cancer Res. 1999 Jan;90(1):69-74.

Growth inhibition of A549 human lung adenocarcinoma cells by L-canavanine is associated with p21/WAF1 induction.

Ding Y, Matsukawa Y, OhtaniFujita N. Department of Preventive Medicine, Kyoto Prefectural University of Medicine.

L-Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L-arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin-dependent kinase inhibitor, p21/WAF1. The p53-dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV-mediated G1 phase arrest.

Shock. 1996 Sep;6(3):194-200.

Effects of L-canavanine, an inhibitor of inducible nitric oxide synthase, on endotoxin mediated shock in rats.

Fatehi-Hassanabad Z, Burns H, Aughey EA. Department of Physiology and Pharmacology, University of Strathclyde, Scotland.

The effects of L-canavanine, an inhibitor of nitric oxide synthase, on endotoxin-induced shock was investigated in the pentobarbitone anesthetized rat. Endotoxin infusion (2.5 mg kg-1 h-1 over 6 h) produced progressive and marked hypotension and hypoglycemia. Electron microscopy showed marked changes in the kidney, comprising severe endothelial cell disruption and the accumulation of platelets in the blood vessels. In the lung, there was marked accumulation of polymorphonuclear leukocytes in small blood vessels and endothelial disruption. Treatment with L-canavanine (10 mg kg-1 by bolus injection each hour starting 70 min after endotoxin or saline infusion) significantly reduced endotoxin-induced hypotension, without any effect on the hypoglycemia. This treatment markedly reduced the endotoxin-induced electron microscopical changes in the kidneys and lungs. Although L-canavanine, like L-NAME, inhibited both cerebellar constitute and splenic inducible nitric oxide synthase in vitro, in contrast to L-NAME it did not modify either arterial blood pressure or carotid artery blood flow in control rats. The data are consistent with L-canavanine being a selective inhibitor of inducible nitric oxide synthase, at least in vivo, and suggest that inhibitors of this enzyme may be beneficial in endotoxin-induced shock.

Clin Sci (Lond). 1996 May;90(5):369-77.

Beneficial effects of L-canavanine, a selective inhibitor of inducible nitric oxide synthase, during rodent endotoxaemia.

Liaudet L, Feihl F, Rosselet A. Institute of Pathophysiology, University Hospital, Lausanne, Switzerland.

1. The cardiovascular failure in sepsis may result from increased nitric oxide biosynthesis, through the diffuse expression of an inducible nitric oxide synthase. In such conditions, nitric oxide synthase inhibitors might be of therapeutic value, but detrimental side effects have been reported with their use, possibly related to the blockade of constitutive nitric oxide synthase. Therefore, the use of selective inhibitors of inducible nitric oxide synthase might be more suitable. The aim of this study was to evaluate the effects of L-canavanine, a potentially selective inhibitor of inducible nitric oxide synthase, in an animal model of septic shock. 2. Anaesthetized rats were challenged with 10 mg/kg lipopolysaccharide intravenously. One hour later, they randomly received a 5 h infusion of either L-canavanine (20 mg h-1 kg-1, n = 15), nitro-L-arginine methyl ester (5 mg h-1 kg-1, n = 13) or 0.9% NaCl (2 ml h-1 kg-1, n = 21). Lipopolysaccharide induced a progressive fall in blood pressure and cardiac index, accompanied by a significant lactic acidosis and a marked rise in plasma nitrate. All these changes were significantly attenuated by L-canavanine, which also improved the tolerance of endotoxaemic animals to acute episodes of hypovolaemia. In addition, L-canavanine significantly increased survival of mice challenged with a lethal dose of lipopolysaccharide. In contrast to L-canavanine, nitro-L-arginine methyl ester increased blood pressure at the expense of a severe fall in cardiac index, while largely enhancing lactic acidosis. This agent did not improve survival of endotoxaemic mice. In additional experiments, we found that the pressor effect of L-canavanine in advanced endotoxaemia (4 h) was reversed by L-arginine, confirming that it was related to nitric oxide synthase inhibition. In contrast, L-canavanine did not exert any influence on blood pressure in the very early stage (first hour) of endotoxaemia or in the absence of lipopolysaccharide exposure, indicating a lack of constitutive nitric oxide synthase inhibition by this agent. 3. In conclusion, L-canavanine produced beneficial haemodynamic and metabolic effects and improved survival in rodent endotoxic shock. The actions of L-canavanine were associated with a selective inhibition of inducible nitric oxide synthase and were in marked contrast to the deleterious consequences of nitro-L-arginine methyl ester, a non-selective nitric oxide synthase inhibitor, in similar conditions.

Eur J Pharmacol. 1996 Jan 11;295(2-3):215-20.

Inhibition of nitric oxide formation with L-canavanine attenuates endotoxin-induced vascular hyporeactivity in the rat.

Cai M, Sakamoto A, Ogawa R. Department of Anaesthesiology, Nippon Medical School, Tokyo, Japan.

L-Canavanine, a selective inhibitor of inducible nitric oxide (NO) synthase, has beneficial effects on the circulatory failure of rats with endotoxin shock. To investigate the direct relationship between these beneficial effects and the inhibition of the formation of NO in response to L-canavanine in endotoxin shock in the rat, we detected changes in venous nitrosyl-hemoglobin (NO-hemoglobin) levels using an electron spin resonance (ESR) assay. Anaesthetized rats were injected with lipopolysaccharide (10 mg/kg i.v.). 1 h after the lipopolysaccharide injection, the rats were divided into four groups: a lipopolysaccharide group receiving 0.3 ml of saline hourly, an L-canavanine 10 or an L-canavanine 20 group receiving L-canavanine 10 or 20 mg/kg i.v. hourly, respectively, and an L-NAME group receiving NG-nitro-L-arginine methyl ester (L-NAME) 15 mg/kg followed by 10 mg/kg i.v. hourly. A sham group received saline instead of lipopolysaccharide, and an L-canavanine group received L-canavanine 20 mg/kg i.v. hourly, 1 h after the saline injection. At 5 h after the lipopolysaccharide or saline injection, pressor responses to noradrenaline (1 microgram/kg i.v.) were obtained. In the lipopolysaccharide group, lipopolysaccharide caused a progressive decrease in mean arterial pressure and an impairment of pressor responsiveness to noradrenaline. Administration of L-canavanine or L-NAME attenuated the endotoxin-induced hypotension and vascular hyporeactivity to noradrenaline. L-Canavanine did not alter mean arterial pressure and the pressor response to noradrenaline in the L-canavanine group. The endotoxin-induced increases in venous levels of NO-hemoglobin were significantly inhibited by L-canavanine or L-NAME. These data indicate that the beneficial hemodynamic effects of L-canavanine are associated with inhibition of the enhanced formation of NO by inducible NO synthase in a rat model of endotoxin shock. L-Canavanine is a potential agent in the treatment of endotoxin shock.

Anticancer Drugs. 1995 Aug;6(4):586-93.

Combination therapy with 5-fluorouracil and L-canavanine: in vitro and in vivo studies.

Swaffar DS, Ang CY. Division of Pharmacology and Toxicology, School of Pharmacy, Northeast Louisiana University, Monroe, USA.

L-Canavanine (CAV) is a potent L-arginine antagonist, produced by legumes such as the jack bean, Canavalia ensiformis. CAV is cytotoxic to MIA PaCa-2 human pancreatic cancer cells. We sought to determine whether CAV's efficacy as an anticancer agent might be increased in combination with 5-fluorouracil (5-FU), a pyrimidine antimetabolite with activity against solid tumors. Using optimal conditions for the expression of CAV's cytotoxicity against MIA PaCa-2 cells, CAV was more cytotoxic to the cells than 5-FU. The combination of both drugs at a fixed molar ratio of 1:1 exhibited synergistic effects in the cells as determined by combination index analysis. The combination of 5-FU:CAV was tested at a ratio of 5:1 and exhibited antagonism at lower effect levels, additivity at 50% effect levels and slight synergism at higher effect levels. A 10:1 combination of both drugs (5-FU:CAV) exhibited antagonistic effects at all levels. When the drugs were combined at a molar ratio of 20:1, increased antagonism was observed. When CAV (1.0 or 2.0 g/kg daily) and/or 5-FU (35 mg/kg daily) was administered to colonic tumor-bearing rats for five consecutive days, the antitumor activity of the drug combination was significantly greater than the combined effects of either drug alone. However, the body weight loss experienced by CAV-treated rats was increased in those rats exposed to a combination of both drugs. These studies using different tumors provide in vitro and in vivo evidence that combination therapy offers a viable means of improving CAV's intrinsic efficacy while decreasing the concentration of 5-FU required to produce the same cytotoxic effect.

Eur J Pharmacol. 1994 Dec 12;271(1):87-92.

L-canavanine restores blood pressure in a rat model of endotoxic shock.

Teale DM, Atkinson AM. Zeneca Pharmaceuticals, Department of Infection Research, Macclesfield, Cheshire, UK.

Administration of lipopolysaccharide to anaesthetised rats produced a reduction in mean arterial pressure, an increase in heart rate, and death at 4-6 h. Intravenous infusion of NG-nitro-L-arginine methyl ester (50 mg/kg), an inhibitor of constitutive and inducible nitric oxide (NO) synthase, 60 min after challenge with lipopolysaccharide, caused an immediate increase in blood pressure followed by a precipitous fall in pressure, and death. In contrast, intravenous infusion of L-canavanine (100 mg/kg), reported to be a selective inhibitor of inducible NO synthase in vitro, 60 min and 180 min after lipopolysaccharide challenge, produced an increase in mean arterial pressure and reversed the lipopolysaccharide induced hypotension. However, in lipopolysaccharide challenged animals protected from hypotension by administration of L-canavanine (60 min post challenge), intravenous infusion of NG-nitro-L-arginine methyl ester at 180 min post challenge caused an immediate rise in mean arterial pressure, followed by a rapid fall in blood pressure and heart rate, and sudden death. In contrast, a second dose of L-canavanine at 180 min post challenge maintained blood pressure for the duration of the experiment. These findings indicate that inhibition of both constitutive and inducible NO synthase during endotoxaemia is lethal. However, the use of a selective inhibitor of inducible NO synthase restores mean arterial pressure to baseline, and offers a therapeutic approach to managing hypotension in shock.

Cancer Res. 1994 Dec 1;54(23):6045-8.

Inhibition of the growth of human pancreatic cancer cells by the arginine antimetabolite L-canavanine.

Swaffar DS, Ang CY, Desai PB. Division of Pharmacology and Toxicology, Northeast Louisiana University, Monroe, USA.

L-Canavanine (CAV), the L-2-amino-4-guanidinooxy structural analogue of L-arginine (ARG), is a potent ARG antagonist which occurs in the jack bean, Canavalia ensiformis. This ARG antimetabolite is active against L1210 murine leukemia and a solid colonic tumor in the rat. Our initial studies using a microtiter assay show that CAV exhibits a 50% inhibitory concentration of approximately 2 mM against the human pancreatic adenocarcinoma cell line, MIA PaCa-2, when these cells are grown in Dulbecco's modified Eagle's medium containing 0.4 mM ARG. When the ARG concentration is reduced to 0.4 microM, the 50% inhibitory concentration for CAV falls precipitously to 0.01 mM. The pronounced increase in the ability of CAV to inhibit MIA PaCa-2 cell growth at the lower ARG concentration may result from enhanced CAV competition with ARG for incorporation into newly synthesized cellular proteins. At 0.4 microM ARG, 30 mM CAV almost completely inhibits cell growth by 6 h. In contrast, with 0.4 mM ARG, complete inhibition does not occur until after 48 h. A dramatic reversal of growth inhibition caused by a very high concentration of CAV was observed when cells treated with CAV were replenished with a high concentration of ARG. Our results suggest that CAV has real potential as a lead compound for the development of analogues with enhanced activity against human pancreatic cancer.

Anticancer Res. 1992 May-Jun;12(3):757-62.

Stress response, survival and enhancement of heat sensitivity in a human melanoma cell line treated with L-canavanine.

Mattei E, Damasi D, Mileo AM, Delpino A, Ferrini U.
Istituto Tecnologie Biomediche, C.N.R., Rome, Italy.

L-Canavanine, like other aminoacid analogs, induces the synthesis of heat shock proteins (HPSs) but, unlike heat or other stressing agents, it fails to induce thermotolerance. We have studied the synthesis and the intracellular distribution of HSPs induced by canavanine, the effects of this analog on the viability and thermal sensitivity of a human melanoma cell line (M14) and the capacity of canavanine-induced HSPs to self regulate their own synthesis. Evidence indicates that the HSP induction is time--and dose--dependent and, also in the presence of arginine, is not associated with the development of thermotolerance. On the contrary, cells become more heat sensitive and are less efficient in the control of the feed-back mechanism that regulates HSP synthesis. The possible utilization of this substance as a potential aid for the treatment of tumors, in association with heat, was examined.

Clin Immunol Immunopathol. 1990 Apr;55(1):97-108.

L-canavanine acts on suppressor-inducer T cells to regulate antibody synthesis: lymphocytes of systemic lupus erythematosus patients are specifically unresponsive to L-canavanine.

Morimoto I, Shiozawa S, Tanaka Y. Department of Medicine, Kobe University School of Medicine, Japan.

L-Canavanine (LC) is an amino acid contained in alfalfa seeds that provokes a disease state similar to systemic lupus erythematosus (SLE) in primates. In vitro experiments showed that LC stimulated proliferation of human phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) and T cells of healthy donors but not of pokeweed mitogen (PWM)-stimulated PBMC. LC inhibited spontaneous generation of immunoglobulin-secreting cells (ISC) of PBMC, while it enhanced ISC generation of CD8(-) cells. LC inhibited PWM-induced ISC generation of CD8(-) cells but not of CD4(-) cells, indicating that LC stimulates CD8(-) cells more strongly than CD4(-) cells. The stimulation index of lymphocyte proliferation (PHA + LC/PHA) was greater in CD8(-)Leu8(+) cells than CD8(-)Leu8(-) cells. The stimulation index was also higher in PBMC than in PBMC plus CD8(-)Leu8(-) cells, the former population containing relatively increased CD8(-)Leu8(+) cells. These findings suggest that LC acts mainly on CD8(-)Leu8(+) cells. That LC acts on CD8(-)Leu8(+) cells was confirmed by the finding that LC inhibited ISC generation of non-T plus CD4(+)Leu8(+), but not of non-T plus CD8(-)Leu8(-) cells. In addition, we found that PBMC of SLE patients were specifically unresponsive to LC stimulation. The stimulation index of lymphocyte proliferation (PHA + LC/PHA) in SLE patients (n = 16) was 0.97 +/- 0.19, whereas that in age-matched healthy control (n = 17) was 1.45 +/- 0.40 (P less than 0.001). Patients with active disease were especially unresponsive to LC. Its responsiveness did not correlate with the dose of prednisolone administered. These findings suggest that the lymphocyte response to LC depends primarily on the existence of functional CD8(-)Leu8(+) cells. Moreover, it appears that suppressor-inducer T cells, responsive to LC, are especially deficient in SLE.

Kobe J Med Sci. 1989 Dec;35(5-6):287-98.

A study on immunological effects of L-canavanine.

Morimoto I.

L-canavanine (LC) is an amino acid contained in alfalfa seeds that provokes a disease state similar to systemic lupus erythematosus (SLE) in primates. In vitro experiments show that LC mainly acts on CD8(-)Leu8(+) T cells to regulate antibody synthesis and lymphocyte proliferation. The lymphocytes of SLE patients were poorly responsive to LC stimulation, suggesting that CD8(-)Leu8(+) T cells are specifically deficient in SLE. Although the precise mechanism of intracellular action of LC is not yet clear, we found that the intracellular calcium level [( Ca2+]i) increased in response to LC. This increase in [Ca2+]i may be partially responsible for the mechanism of action of LC on lymphocytes.

J Biol Chem. 1989 Aug 15;264(23):13693-6.

L-canavanine incorporation into vitellogenin and macromolecular conformation.

Rosenthal GA, Reichhart JM, Hoffmann JA. T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, USA.

L-Canavanine is a potentially deleterious arginine antimetabolite whose toxicity is expressed in canavanine-sensitive organisms ranging from viruses to humans. Canavanine, a substrate for arginyl-tRNA synthetase, is incorporated into nascent polypeptide chains in place of arginine. This substitution results in the production of structurally aberrant, canavanyl proteins. Chemical, physical, and immunological studies of native and canavanine-containing vitellogenin obtained from female migratory locusts (Locusta migratoria migratorioides (Orthoptera] provide the first experimental evidence that canavanine can disrupt the tertiary and/or quaternary structure that yields the three-dimensional conformation unique to the protein. These findings enhance our understanding of the biochemical basis for canavanine's antimetabolic and potent insecticidal properties.

Cancer Res. 1983 Sep;43(9):4180-2.

Enhancement of human tumor cell killing by L-canavanine in combination with gamma-radiation.

Green MH, Ward JF.

On the basis of several physiological properties of L-canavanine, we have tested the prediction that this analogue of arginine would enhance the cytotoxic effects of gamma-rays in mammalian cells. Using the human colonic tumor cell line, HT-29, time-dose studies were performed with log-phase cultures in order to determine conditions which maximize the incorporation of L-canavanine into cellular proteins while leaving a large fraction of the cells viable for subsequent gamma-ray survival measurements. At an input ratio of 2.5 (L-canavanine:arginine), the analogue exerted a cytostatic effect on the cells for at least 6 days following one cell division. Little cell killing (less than 20%) by clonogenicity was caused by L-canavanine during the first 12 hr of treatment of log-phase cells, even at a L-canavanine:arginine ratio of 20. A 24-hr exposure, however, produced an exponential decrease in survival as a function of L-canavanine concentration. The interaction between L-canavanine treatment and gamma-ray damage with respect to cell survival was examined under several conditions and times based on the above findings. Optimal enhancement of X-ray-induced cytotoxicity (assayed by loss of clonogenicity) was observed with a 48-hr exposure to the analogue at a L-canavanine:arginine ratio of 10. A marked increase in radiosensitivity was observed when L-canavanine was administered either before or after irradiation of the cells. In both protocols, enhancement was seen at all radiation doses. Together with our earlier findings showing the antitumor activity of L-canavanine in L1210 murine leukemia, these results suggest the potential usefulness of this amino acid analogue in the treatment of cancer.

J Ultrastruct Res. 1983 Mar;82(3):241-63.

Nuclear alterations induced by cadmium chloride and L-canavanine in HeLa S3 cells. Accumulation of perichromatin granules.

Cervera J, Alamar M, Martinez A, Renau-Piqueras J.

The effects of L-canavanine and cadmium on the ribonucleoprotein constituents of HeLa S3 cells have been analyzed. Both chemicals induce a similar pattern of alterations in different RNP structures as well as in both RNA and protein synthesis. Pulse and chase autoradiographic experiments reveal that both canavanine and cadmium induce a preferential inhibition of nucleolar RNA synthesis and a slowdown in the transport or processing of nucleolar and extranucleolar RNA. Nucleoli become round and compact. Accumulation of perichromatin granules and fibrils occurs, there is a depletion of interchromatin fibrils, and nuclear formations appear which seem to be involved in the morphogenesis of perichromatin granules accumulated during the treatments. The appearance of clusters of 29- to 35-nm granules might be related with a deficient assembling of constituents of perichromatin granules. The effects of different inhibitors of the transcriptional processes on the accumulation of perichromatin granules suggest that these granules represent a particular subpopulation of hnRNP.

Anticancer Res. 1981;1(5):275-7.

Cytotoxicity of L-canavanine in vitro.

Naha PM.

Cytotoxicity of L-canavanive, a structural analogue of L-arginine, was tested by means of (1) inhibition of DNA synthesis in Balb/c-3T3 cell line and (2) inhibition of mitosis in peripheral blood lymphocytes in culture. The cytostatic nature of inhibition caused by L-canavanine was indicated by the complete recovery of cells after withdrawal of the drug.

Cancer Res. 1980 Mar;40(3):535-7.

Antitumor activity of L-canavanine against L1210 murine leukemia.

Green MH, Brooks TL, Mendelsohn J, Howell SB.

We have made a preliminary assessment of the antitumor activity of the arginine analog, L-canavanine, in leukemic mice. This analog is known to substitute for arginine in protein biosynthesis in many prokaryotic and eukaryotic systems. Previous studies with cells grown in vitro indicated that canavanine caused a marked inhibition of DNA synthesis and viability. The system used in the present study was C57BL/6 x DBA/2 F mice bearing L1210 leukemic cells. Following an i.v. injection of 10 mg canavanine, the t1/2 beta of canavanine in the serum was estimated at 16 min. This finding suggested that frequent injections of high doses of canavanine would be required for an effect on tumor cell proliferation. DNA synthesis by the L1210 cells, assayed by [3H]thymidine incorporation, fell to 9% of the control value after 12 hourly i.p. injections of canavanine (20 mg each). A constant s.c. infusion of 20 mg/hr for 24 hr caused an 86% inhibition of DNA synthesis. The antitumor activity of canavanine was tested against L1210, using a 24-hr infusion schedule with treatment starting 24 hr after i.p. inoculation of 10(5) cells. An optimal dose of 18 g/kg body weight produced a median increased lifespan of 44% (p less than 0.005). These results suggest that L-canavanine may be useful as an antitumor agent.

Q Rev Biol. 1977 Jun;52(2):155-78.

The biological effects and mode of action of L-canavanine, a structural analogue of L-arginine.

Rosenthal GA.

Many of the 200 or so non-protein amino acids synthesized by higher plants are related structurally to the constituents of common proteins. L-Canavanine, the guanidinooxy structural analogue of L-arginine, is representative of this group. It has provided valuable insight into the biological effects and the mode of action of non-protein amino acids which acts as analogues of the protein amino acids. The arginyl-tRNA synthetases of numerous canavanine-free species charge canavanine, and canavanine is subsequently incorporated into the nascent polypeptide chain. Production of canavanine-containing proteins ultimately can disrupt critical reactions of RNA and DNA metabolism as well as protein synthesis. Canavanine also affects regulatory and catalytic reactions of arginine metabolism, arginine uptake, formation of structural components, and other cellular precesses. In these ways, canavanine alters essential biochemical reactions and becomes a potent antimetabolite of arginine in a wide spectrum of species.

Arthritis Rheum. 1985 Jan;28(1):52-7.

Effects of L-canavanine on T cells may explain the induction of systemic lupus erythematosus by alfalfa.

Alcocer-Varela J, Iglesias A, Llorente L, Alarcon-Segovia D.
Alfalfa sprouts can induce systemic lupus erythematosus (SLE) in monkeys. This property of alfalfa sprouts has been attributed to their non-protein amino acid constituent, L-canavanine. Occurrence of autoimmune hemolytic anemia and exacerbation of SLE have been linked to ingestion of alfalfa tablets containing L-canavanine. In this report we show that L-canavanine has dose-related effects in vitro on human immunoregulatory cells, which could explain its lupus-inducing potential. These effects include: 1) diminution of the mitogenic response to both phytohemagglutinin and concanavalin A but not to pokeweed mitogen, as determined in both thymidine incorporation and cell cycle studies, and 2) abrogation of concanavalin A-induced suppressor cell function, which results in increased release of both IgG and DNA binding activity into supernatants by cells from normal subjects and SLE patients. These immunoregulatory effects of L-canavanine may explain the induction or exacerbation of SLE by alfalfa.

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