Deuterium labeling improves the therapeutic index of 3,3'-diselenodipropionic acid as an anticancer agent: insights from redox reactions.

3,3'-Diselenodipropionic acid (DSePA), a selenocystine derivative, has been previously reported as an oral supplement for anticancer/radio-modulation activities. The present study is focused on devising a strategy to synthesize and characterize the deuterated derivative of DSePA and on understanding the effect of deuteration on its therapeutic index by comparing its cytotoxicity in cancerous versus non-cancerous cell types. In this context, the synthesis of 3,3'-diselenodipropionic acid-D8 (D-DSePA) was accomplished in ∼42% yield. Further, the results clearly established that the deuteration of DSePA significantly reduced its cytotoxicity in non-cancerous cell types while retaining its cytotoxicity in cancerous cell lines. Together, D-DSePA displayed a ∼5-fold higher therapeutic index than the non-deuterated derivative for anticancer activity. The biochemical and NMR studies confirmed that the better biocompatibility of D-DSePA than its non-deuterated derivative in non-cancerous cells was due to its ability to undergo slower redox reactions and to cause lesser inhibition of intracellular redox enzymes.

3,3'-Diselenodipropionic acid (DSePA) induces reductive stress in A549 cells triggering p53-independent apoptosis: A novel mechanism for diselenides.

The aim of present study was to investigate the anticancer mechanisms of 3,3'-diselenodipropionic acid (DSePA), a redox-active organodiselenide in human lung cancer cells. DSePA elicited a significant concentration and time-dependent cytotoxicity in human lung cancer cell line A549 than in normal WI38 cells. The cytotoxic effect of DSePA was preceded by an acute decrease in the level of basal reactive oxygen species (ROS) and a concurrent increase in levels of reducing equivalents (like GSH/GSSG and NADH/NAD) within cells. Further, a series of experiments were performed to measure the markers of intrinsic (Bax, cytochrome c and caspase-9), extrinsic (TNFR, FADR and caspase-8) and endoplasmic reticulum (ER) stress (protein ubiquitylation, calcium flux, Bip, CHOP and caspase-12) pathways in DSePA treated cells. DSePA treatment significantly increased the levels of all the above markers. Moreover, DSePA did not alter the expression and phosphorylation (Ser15) of p53 but caused a significant damage to mitochondria. Pharmacological modulation of GSH level by BSO and NAC in DSePA treated cells led to partial abrogation and augmentation of cell kill respectively. This established the role of reductive stress as a trigger for the apoptosis induced by DSePA treatment. Finally, in vitro anticancer activity of DSePA was also corroborated by its in vivo efficacy of suppressing the growth of A549 derived xenograft tumor in SCID mice. In conclusion, above results suggest that DSePA induces apoptosis in a p53 independent manner by involving extrinsic and intrinsic pathways together with ER stress which can an interesting strategy for lung cancer therapy.

2,2'-Dipyridyl diselenide (Py2Se2) induces G1 arrest and apoptosis in human lung carcinoma (A549) cells through ROS scavenging and reductive stress.

Organo-diselenides are well documented for pro-oxidant effects in tumor cells. However, the present study demonstrated that 2,2'-dipyridyl diselenide (Py2Se2) induced cytotoxicity in human non-small cell lung carcinoma (A549) cells through reductive stress marked by a significant decrease in the basal level of reactive oxygen species and a concurrent decrease in the ratio of oxidised (GSSG) and reduced (GSH) glutathione. The IC50 (concentration inducing 50% cytotoxicity) of Py2Se2 in A549 and human normal lung fibroblast (WI38) cells was ∼8.5 µM and ∼5.5 µM, respectively, indicating that Py2Se2 did not exhibit selective toxicity towards cancer cells. Cell free studies indicated that Py2Se2 acted as a substrate of thioredoxin reductase (TrxR) and accordingly it was proposed that TrxR mediated reduction of Py2Se2 within cells might be generating intermediates leading to a reductive environment. Despite generating a reducing environment, Py2Se2 caused significant DNA damage, G1 phase arrest and apoptosis. The mechanistic investigation revealed that Py2Se2 induced G1 arrest was mediated through up-regulation of p21 transcript in a p53 independent manner. Further, the apoptotic effect of Py2Se2 was associated with the increase in the levels of unfolded protein response markers like BiP and CHOP, mitochondrial permeability (JC1) and apoptotic markers such as cleaved caspase-3 and poly (ADP-ribose) polymerase. Finally, pre-treatment with N-acetylcysteine (a stimulator of GSH biosynthesis) or l-buthionine sulfoximine (an inhibitor of GSH biosynthesis) increased and decreased the Py2Se2 mediated apoptosis, respectively. This confirmed that the cytotoxicity of Py2Se2 in A549 cells was triggered through reductive stress.

Oral administration of 3,3'-diselenodipropionic acid prevents thoracic radiation induced pneumonitis in mice by suppressing NF-kB/IL-17/G-CSF/neutrophil axis.

The incidence of symptomatic radiation induced lung pneumonitis (RILP), a major dose limiting side effect of thoracic radiotherapy, is in the range of 15-40%. Therapeutic options for the prevention and treatment of RILP are limited. Hence there is a need for developing novel radioprotectors to prevent RILP which can be patient compliant. This study sought to evaluate the efficacy of oral 3,3'-diselenodipropionic acid (DSePA), a novel selenocystine derivative to prevent RILP. C3H/HeJ (pneumonitis responding) mice received a single dose of 18 Gy, whole thorax irradiation and a subset were treated with DSePA orally (2.5 mg/kg), three times per week beginning 2 h post irradiation and continued till 6 months. DSePA delayed onset of grade ≥ 2 RILP by 45 days compared to radiation control (~105 versus ~60 days). It also reversed the severity of pneumonitis in 3/10 radiation treated mice leading to significant improvement in asymptomatic survival compared to radiation control (~180 versus ~102 days). DSePA significantly (p < 0.05) reduced the radiation-mediated infiltration of polymorphonuclear neutrophils (PMN) and elevation in levels of cytokines such as IL1-ß, ICAM-1, E-selectin, IL-17 and TGF-ß in the bronchoalveolar lavage fluid. Moreover DSePA lowered PMN-induced oxidants, maintained glutathione peroxidase activity and suppressed NF-kB/IL-17/G-CSF/neutrophil axis in the lung of irradiated mice. Additionally, this compound did not protect A549 (lung cancer) derived xenograft tumor from radiation exposure in SCID mice. DSePA offers protection to normal lung against RILP without affecting radiation sensitivity of tumors. It has the potential to be developed as an oral agent for preventing RILP.

Pharmacodynamics and proteomic analysis of acalabrutinib therapy: similarity of on-target effects to ibrutinib and rationale for combination therapy.

Acalabrutinib, a highly selective Bruton's tyrosine kinase inhibitor, is associated with high overall response rates and durable remission in previously treated chronic lymphocytic leukemia (CLL); however, complete remissions were limited. To elucidate on-target and pharmacodynamic effects of acalabrutinib, we evaluated several laboratory endpoints, including proteomic changes, chemokine modulation and impact on cell migration. Pharmacological profiling of samples from acalabrutinib-treated CLL patients was used to identify strategies for achieving deeper responses, and to identify additive/synergistic combination regimens. Peripheral blood samples from 21 patients with relapsed/refractory CLL in acalabrutinib phase I (100-400 mg/day) and II (100 mg BID) clinical trials were collected prior to and on days 8 and 28 after treatment initiation and evaluated for plasma chemokines, reverse phase protein array, immunoblotting and pseudoemperipolesis. The on-target pharmacodynamic profile of acalabrutinib in CLL lymphocytes was comparable to ibrutinib in measures of acalabrutinib-mediated changes in CCL3/CCL4 chemokine production, migration assays and changes in B-cell receptor signaling pathway proteins and other downstream survival proteins. Among several CLL-targeted agents, venetoclax, when combined with acalabrutinib, showed optimal complementary activity in vitro, ex vivo and in vivo in TCL-1 adoptive transfer mouse model system of CLL. These findings support selective targeting and combinatorial potential of acalabrutinib.

Duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocytic leukemia cells to venetoclax (ABT-199).

Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma. In CLL, duvelisib monotherapy is associated with high iwCLL (International Workshop on Chronic Lymphocytic Leukemia) and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene expression studies (RNAseq, Nanostring, Affymetrix array and real-time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex vivo-cultured CLL cells obtained from patients on duvelisib compared with pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

The phosphoinositide-3-kinase (PI3K)-delta and gamma inhibitor, IPI-145 (Duvelisib), overcomes signals from the PI3K/AKT/S6 pathway and promotes apoptosis in CLL.

The functional relevance of the B-cell receptor (BCR) and the evolution of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B-cell malignancies. Inhibition of p110δ with idelalisib has shown clinical activity in chronic lymphocytic leukemia (CLL). The dynamic interplay of isoforms p110δ and p110γ in leukocytes support the hypothesis that dual blockade may provide a therapeutic benefit. IPI-145, an oral inhibitor of p110δ and p110γ isoforms, sensitizes BCR-stimulated and/or stromal co-cultured primary CLL cells to apoptosis (median 20%, n=57; P<0.0001) including samples with poor prognostic markers, unmutated IgVH (n=28) and prior treatment (n=15; P<0.0001). IPI-145 potently inhibits the CD40L/IL-2/IL-10 induced proliferation of CLL cells with an IC50 in sub-nanomolar range. A corresponding dose-responsive inhibition of pAKT(Ser473) is observed with an IC50 of 0.36 nM. IPI-145 diminishes the BCR-induced chemokines CCL3 and CCL4 secretion to 17% and 37%, respectively. Pre-treatment with 1 µM IPI-145 inhibits the chemotaxis toward CXCL12; reduces pseudoemperipolesis to median 50%, inferring its ability to interfere with homing capabilities of CLL cells. BCR-activated signaling proteins AKT(Ser473), BAD(Ser112), ERK(Thr202/Tyr204) and S6(Ser235/236) are mitigated by IPI-145. Importantly, for clinical development in hematological malignancies, IPI-145 is selective to CLL B cells, sparing normal B- and T-lymphocytes.

Recurrent cholangitis in the tropics: worm or cast?

The development of biliary casts is very rare, especially in non-liver transplant patients. The etiology of these casts is uncertain but several factors have been proposed which lead to bile stasis and/or gallbladder hypo-contractility and promote cast formation. Here, we report a 54-year-old male, with diabetes and ischemic heart disease, who presented with recurrent attacks of cholangitis. Magnetic resonance cholangiopancreatography revealed linear T1 hyperintense and T2 hypointense filling defects in the right and left hepatic ducts extending into the common hepatic duct, and a calculus in the lower common bile duct, raising a suspicion of worm in the biliary tree. In view of failed attempts at extraction on endoscopy, patient underwent surgery. At exploration, biliary casts and stones were extracted from the proximal and the second order bile ducts, with the help of intraoperative choledochoscopy and a bilio-enteric anastomosis was accomplished. Although endoscopic retrieval of the biliary cast can be employed as first-line management, surgery should be considered in case it fails.

Distribution of small integrin-binding ligand, N-linked glycoproteins (SIBLING) in the condylar cartilage of rat mandible.

The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of non-collagenous proteins closely related to osteogenesis. In this study, the authors systematically evaluated the presence and distribution of four SIBLING family members, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (OPN), in rat mandibular condylar cartilage using protein chemistry and immunohistochemistry. For protein chemistry, SIBLING proteins in the dissected condylar cartilage were extracted with 4M guanidium-HCl, separated by ion-exchange chromatography, and analyzed by Western immunoblotting. Immunohistochemistry was employed to assess the distribution of these four SIBLING proteins in the condylar cartilage of 2-, 5- and 8-week-old rats. Results from both approaches showed that all four members are expressed in the condylar cartilage. DSPP, unlike that observed in dentin and bone, exists as a full-length form (uncleaved) in the condylar cartilage. The NH(2)-terminal fragment of DMP1 is mainly detected in the matrix of the cartilage while the COOH-terminal fragment is primarily localized in the nuclei of cells in the chondroblastic and hypertrophic layers. The data obtained in this investigation provide clues about the potential roles of these SIBLING proteins in chondrogenesis.

Combination of cladribine and cytarabine is effective for childhood acute myeloid leukemia: results of the St Jude AML97 trial.

Because cladribine can increase cytarabine triphosphate levels, we tested a cladribine-cytarabine combination in the St Jude AML97, trial in which this combination was administered before standard chemotherapy to 96 children with acute myeloid leukemia (AML) or myelodysplastic syndrome. Patients received a 5-day course of cladribine (9 mg/m(2) per dose) and cytarabine either as daily 2-h infusions (500 mg/m(2) per dose) (arm A) or a continuous infusion (500 mg/m(2) per day) (arm B). Ara-CTP levels and inhibition of DNA synthesis increased from day 1 to day 2, but were not different between the two arms. In addition, the median blast percentages at day 15 did not differ between arms A and B, but patients treated in arm A had shorter intervals between the initiation of the first and second courses of therapy. Thus, although there were trends toward better complete remission rates and overall survival for patients treated in arm B, the reduced efficacy of arm A may have been partially compensated by more intense timing of therapy for that group. For all patients, 5-year event-free survival and overall survival estimates were 44.1+/-5.4 and 50.0+/-5.5%. Our results suggest that cladribine in combination with continuous-infusion cytarabine is effective therapy for childhood AML.

Phase I study of 506U78 administered on a consecutive 5-day schedule in children and adults with refractory hematologic malignancies.

PURPOSE: A phase I study was conducted to determine the maximum-tolerated dose (MTD), toxicity profile, and pharmacokinetics of a novel purine nucleoside, nelarabine, a soluble prodrug of 9-beta-D-arabinosylguanine (araG; Nelarabine), in pediatric and adult patients with refractory hematologic malignancies. PATIENTS AND METHODS: Between April 1994 and April 1997, 93 patients with refractory hematologic malignancies were treated with one to 16 cycles of study drug. Nelarabine was administered daily, as a 1-hour intravenous infusion for 5 consecutive days, every 21 to 28 days. First-cycle pharmacokinetic data, including plasma nelarabine and araG levels, were obtained on all patients treated. Intracellular phosphorylation of araG was studied in samples of leukemic blasts from selected patients. RESULTS: The MTDs were defined at 60 mg/kg/dose and 40 mg/kg/dose daily x 5 days in children and adults, respectively. Dose-limiting toxicity (DLT) was neurologic in both children and adults. Myelosuppression and other significant organ toxicities did not occur. Pharmacokinetic parameters were similar in children and adults. Accumulation of araGTP in leukemic blasts was correlated with cytotoxic activity. The overall response rate was 31%. Major responses were seen in patients with T-cell malignancies, with 54% of patients with T-lineage acute lymphoblastic leukemia achieving a complete or partial response after one to two courses of drug. CONCLUSION: Nelarabine is a novel nucleoside with significant cytotoxic activity against malignant T cells. DLT is neurologic. Phase II and III trials in patients with T-cell malignancies are encouraged.

Gemcitabine in hematologic malignancies.

Gemcitabine is a pyrimidine analogue that showed significant activity in solid malignancies. Gemcitabine acts by inhibiting DNA synthesis through chain termination and ribonucleotide reductase inhibition. During initial phase I and II studies, gemcitabine had a low toxicity profile and was well tolerated as a single agent and in combination therapy. Recently, there has been more interest in studying the activity of gemcitabine in hematologic malignancies. Gemcitabine demonstrated good activity in refractory Hodgkin disease patients, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, and acute leukemias. There is a preponderance of evidence on the activity of gemcitabine in vitro in myeloma and leukemic cell lines. The activity of gemcitabine in these disorders will pave the way for incorporating this agent into the early phases of therapy.

Phase II clinical investigation of gemcitabine in advanced soft tissue sarcomas and window evaluation of dose rate on gemcitabine triphosphate accumulation.

PURPOSE: To evaluate the efficacy, toxicity, and optimal dose rate of gemcitabine in adult patients with advanced soft tissue sarcomas (STS) by comparing levels of gemcitabine triphosphate (GTP) in peripheral-blood mononuclear cells (PBMCs) of patients receiving two different dose rates. PATIENTS AND METHODS: Fifty-six assessable patients with STS (17 gastrointestinal [GI] leiomyosarcomas and 39 other histologies) were treated on a two-arm phase II study. Gemcitabine was given at 1 g/m2 as a 30-minute infusion weekly for up to 7 weeks followed by 1 week of rest and reassessment of tumor. Subsequent cycles were given at 1 g/m2 weekly for 3 weeks followed by 1 week of rest. Nine patients underwent cellular pharmacologic studies at two different dose rates (1 g/m2 over a standard 30-minute infusion on week 1 and over pharmacologically based infusion of 150 minutes on week 2) to evaluate GTP levels in PBMCs. RESULTS: Seven partial responses were noted among 39 patients, for an overall response rate of 18% (95% confidence interval, 7% to 29%). Median duration of response was 3.5 months (range, 2 to 13 months). Four of 10 patients with non-GI leiomyosarcomas achieved a partial response. No objective responses were noted in 17 patients with GI leiomyosarcomas. One patient had a mixed response. Median time to progression for all patients (both arms) was 3 months; median survival was 13.9 months. Treatment was generally well tolerated. Comparison of cellular pharmacology demonstrated a significant 1.4-fold increase in the concentration of GTP with the 150-minute infusion. CONCLUSION: Given the limited therapeutic armamentarium for STS, the activity of gemcitabine is encouraging. Its potential for combination therapy in the salvage setting should be studied with pharmacologically guided fixed dose-rate infusion.

8-chloro-cAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma cells.

Previous work with 8-chloro-cAMP (8-Cl-cAMP) has raised questions as to whether it works as a cAMP analogue or as a nucleoside analogue after its conversion to 8-chloro-adenosine (8-Cl-Ado). Although degradation of 8-Cl-cAMP to 8-Cl-Ado in culture medium or plasma has been shown, cellular pharmacology data are missing. The purpose of the present study was to identify the cellular metabolism of these drugs and their actions in a human multiple myeloma cell line. The cells were incubated with either 8-Cl-Ado or 8-Cl-cAMP to follow the cellular metabolism of these agents. Both 8-Cl-cAMP and 8-Cl-Ado incubation resulted in the accumulation of 8-Cl-Ado mono-, di-, and tri-phosphate (8-Cl-ATP), however, the triphosphate was the major cytotoxic metabolite. Accumulation of 8-Cl-ATP was dependent on both the exogenous concentration of 8-Cl-Ado and incubation time. At the 10 microM level of 8-Cl-Ado, >400 microM 8-Cl-ATP accumulated in multiple myeloma cells after continuous incubation for 12 h. Similar incubation with 8-Cl-cAMP also resulted in accumulation of 8-Cl-ATP in the cells, albeit at a lower level. The formation of 8-Cl-ATP from 8-Cl-cAMP was inhibited by >80% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in the medium, suggesting extracellular conversion of 8-Cl-cAMP to 8-Cl-Ado. Cells lacking Ado kinase did not accumulate 8-Cl-ATP, either from 8-Cl-Ado or 8-Cl-cAMP, and were resistant to these agents. There was also a decline in the endogenous level of the cellular ATP pool parallel to the accumulation of 8-C1-ATP. The elimination of 8-Cl-ATP was biphasic and slow from the cells. The accumulation of 8-Cl-ATP and a decline in the ATP pool inhibited RNA synthesis but did not affect DNA synthesis for up to 12 h of incubation. Taken together, these data demonstrate that the cytotoxic metabolite of 8-Cl-Ado and 8-Cl-cAMP is 8-Cl-ATP. Hence, 8-Cl-cAMP serves as a prodrug and is converted to 8-Cl-Ado in medium with subsequent phosphorylation to accumulate as 8-Cl-ATP in cells. At the cellular level, 8-Cl-ATP is associated with a decrease in the endogenous ATP pool; at the nuclear level, it inhibits RNA synthesis.

Evaluation of the combination of nelarabine and fludarabine in leukemias: clinical response, pharmacokinetics, and pharmacodynamics in leukemia cells.

PURPOSE: A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate. PATIENTS AND METHODS: Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days. RESULTS: Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27). CONCLUSION: Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.

High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells.

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125-10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.

Red cell haemolysis test as an in vitro approach for the assessment of toxicity of karanja oil.

The karanja tree grows in parts of India and Australia. The oil from seed kernels was found to be toxic to animals. The annual potential availability of the oil is around 135,000 tons in India. In order to use it for beneficial purposes, it is necessary to detoxify the oil. In the present study, the oil was assessed for toxicity by the red cell haemolysis test and estimating the LDH in the supernatant. The non-lipid constituents were isolated from raw oil by aqueous methanol extraction. The raw oil and the non-lipid fraction were found to haemolyse the red cells with release of LDH, whereas the extracted oil did not show such a manifestation. There was a good correlation between haemolytic activity and LDH released from cells. These findings were further confirmed with in vivo studies where the raw and extracted karanja oils showed 100% and nil mortality in rats dosed orally at 10 and 20 ml/kg body weight, respectively. This haemolysis test can be used as an in vitro method to predict toxicity and to monitor the detoxification of the oils prior to use in in vivo studies for toxicological evaluation. The fatty acid composition of the raw and extracted karanja oils showed no difference.