The Role of Diacylglycerol Kinase in the Amelioration of Diabetic Nephropathy.

The drastic increase in the number of patients with diabetes and its complications is a global issue. Diabetic nephropathy, the leading cause of chronic kidney disease, significantly affects patients' quality of life and medical expenses. Furthermore, there are limited drugs for treating diabetic nephropathy patients. Impaired lipid signaling, especially abnormal protein kinase C (PKC) activation by de novo-synthesized diacylglycerol (DG) under high blood glucose, is one of the causes of diabetic nephropathy. DG kinase (DGK) is an enzyme that phosphorylates DG and generates phosphatidic acid, i.e., DGK can inhibit PKC activation under diabetic conditions. Indeed, it has been proven that DGK activation ameliorates diabetic nephropathy. In this review, we summarize the involvement of PKC and DGK in diabetic nephropathy as therapeutic targets, and its mechanisms, by referring to our recent study.

The neuroprotective function of 2-carba-cyclic phosphatidic acid: Implications for tenascin-C via astrocytes in traumatic brain injury.

We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI.

Antifibrotic effects of 2-carba cyclic phosphatidic acid (2ccPA) in systemic sclerosis: contribution to the novel treatment.

BACKGROUND: Cyclic phosphatidic acid (cPA) has an inhibitory effect on the autotaxin (ATX)/lysophosphatidic acid (LPA) axis, which has been implicated to play an important role in the progression of fibrosis in systemic sclerosis (SSc). The purpose of this study is to assess the antifibrotic activity of cPA for the treatment of SSc using SSc skin fibroblasts and an animal model of bleomycin-induced skin fibrosis. METHODS: We used a chemically stable derivative of cPA (2ccPA). First, we investigated the effect of 2ccPA on extracellular matrix (ECM) expression in skin fibroblasts. Next, the effect of 2ccPA on the intracellular cAMP levels was determined to investigate the mechanisms of the antifibrotic activity of 2ccPA. Finally, we administered 2ccPA to bleomycin-induced SSc model mice to evaluate whether 2ccPA prevented the progression of skin fibrosis. RESULTS: 2ccPA decreased ECM expression in SSc skin fibroblasts and TGF-ß1-treated healthy skin fibroblasts without LPA stimulation. 2ccPA increased the intracellular cAMP levels in skin fibroblasts, suggesting that the antifibrotic effect of 2ccPA was the consequence of the increase in the intracellular cAMP levels. Administration of 2ccPA also ameliorated the progression of bleomycin-induced skin fibrosis in mice. CONCLUSIONS: Our data indicated that 2ccPA had inhibitory effects on the progression of skin fibrosis by abrogating ECM production from activated skin fibroblasts. These cells were repressed, at least in part, by increased intracellular cAMP levels. 2ccPA may be able to be used to treat fibrotic lesions in SSc.

Phosphatidic Acid Enters into the YAP/TAZ Arena.

Reporting in Molecular Cell, Han et al. (2018;2:328-340) show that phosphatidic acid promotes YAP activity through a double-hit inhibition of the LATS kinases, suggesting a therapeutic opportunity for the treatment of YAP/TAZ-dependent cancers and opening new prospects on the connections between lipid signaling and YAP/TAZ biology.

A lecithin phosphatidylserine and phosphatidic acid complex (PAS) reduces symptoms of the premenstrual syndrome (PMS): Results of a randomized, placebo-controlled, double-blind clinical trial.

BACKGROUND & AIMS: Many women experience emotional and physical symptoms around the time of ovulation and more so before menstruation interfering with their daily normal life also known as premenstrual syndrome (PMS). Recent observational data suggest that supplementation with Lipogen's phosphatidylserine (PS) and phosphatidic acid (PA) complex (PAS) alleviates these PMS symptoms. The aim of this study was to confirm these observations on the effects of PAS on PMS symptom severity within a controlled clinical trial setting. METHODS: Forty women aged 18-45 years with a diagnosis of PMS were assigned to either take PAS (containing 400 mg PS & 400 mg PA per day) or a matching placebo. The study comprised 5 on-site visits including 1 baseline menstrual cycle followed by 3 treatment cycles. Treatment intake was controlled for by using an electronic device, the Medication Event Monitoring System (MEMS®). Primary outcome of the study was the PMS symptoms severity as assessed by using the Daily Record of Severity of Problems (DRSP). Further, SIPS questionnaire (a German version of the Premenstrual Symptoms Screening Tool (PSST)), salivary hormone levels (cortisol awakening response (CAR) and evening cortisol levels) as well as serum levels (cortisol, estradiol, progesterone and corticosteroid binding globulin (CBG)) were assessed. RESULTS: PMS symptoms as assessed by the DRSP Total score showed a significantly better improvement (p = 0.001) over a 3 cycles PAS intake as compared to placebo. In addition, PAS treated women reported a greater improvement in physical (p = 0.002) and depressive symptoms (p = 0.068). They also reported a lower reduction of productivity (p = 0.052) and a stronger decrease in interference with relationships with others (p = 0.099) compared to the placebo group. No other DRSP scale or item showed significant results. Likewise, the reduction in the number of subjects fulfilling PMS or premenstrual dysphoric disorder (PMDD) criteria as classified by the SIPS did not differ between the PAS and the placebo group. For the biomarkers, the salivary cortisol percentage increase of the CAR was significantly less pronounced in the follicular phase of cycle 4 than in the follicular phase of cycle 1 for subjects taking PAS when compared to subjects taking placebo (p = 0.018). Furthermore, the change of serum cortisol levels between visit 1 and visit 5 differed significantly between groups (p = 0.043). While serum cortisol levels of PAS treated females slightly decreased between visit 1 and visit 5, cortisol levels of females treated with placebo increased. For all other biomarkers, no treatment effects were observed over the 4 cycles study period. Overall, this study confirms that a daily intake of PAS, containing 400 mg PS and 400 mg PA, can be considered as safe. CONCLUSIONS: Results substantiate the efficacy of PAS in reducing symptoms of PMS. In view of the recent inclusion of severe PMS symptoms (PMDD) in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), the positive results of this clinical study merits consideration of developing the PAS complex as a botanical drug for treatment of PMDD. CLINICAL TRIAL REGISTRATION: The study is registered at Deutsches Register Klinischer Studien with the registration number DRKS00009005.

Protective and therapeutic role of 2-carba-cyclic phosphatidic acid in demyelinating disease.

BACKGROUND: Multiple sclerosis is a neuroinflammatory demyelinating and neurodegenerative disease of the central nervous system characterized by recurrent and progressive demyelination/remyelination cycles, neuroinflammation, oligodendrocyte loss, demyelination, and axonal degeneration. Cyclic phosphatidic acid (cPA) is a natural phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. We reported earlier that cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. We designed, chemically synthesized, and metabolically stabilized derivatives of cPA: 2-carba-cPA (2ccPA), a synthesized compound in which one of the phosphate oxygen molecules is replaced with a methylene group at the sn-2 position. In the present study, we investigated whether 2ccPA exerts protective effects in oligodendrocytes and suppresses pathology in the two most common mouse models of multiple sclerosis. METHODS: To evaluate whether 2ccPA has potential beneficial effects on the pathology of multiple sclerosis, we investigated the effects of 2ccPA on oligodendrocyte cell death in vitro and administrated 2ccPA to mouse models of experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. RESULTS: We demonstrated that 2ccPA suppressed the CoCl2-induced increase in the Bax/Bcl-2 protein expression ratio and phosphorylation levels of p38MAPK and JNK protein. 2ccPA treatment reduced cuprizone-induced demyelination, microglial activation, NLRP3 inflammasome, and motor dysfunction. Furthermore, 2ccPA treatment reduced autoreactive T cells and macrophages, spinal cord injury, and pathological scores in EAE, the autoimmune multiple sclerosis mouse model. CONCLUSIONS: We demonstrated that 2ccPA protected oligodendrocytes via suppression of the mitochondrial apoptosis pathway. Also, we found beneficial effects of 2ccPA in the multiperiod of cuprizone-induced demyelination and the pathology of EAE. These data indicate that 2ccPA may be a promising compound for the development of new drugs to treat demyelinating disease and ameliorate the symptoms of multiple sclerosis.

Multifunctional liposomes delay phenotype progression and prevent memory impairment in a presymptomatic stage mouse model of Alzheimer disease.

The failure of clinical trials largely focused on mild to moderate stages of Alzheimer disease has suggested to the scientific community that the effectiveness of Amyloid-ß (Aß)-centered treatments should be evaluated starting as early as possible, well before irreversible brain damage has occurred. Accordingly, also the preclinical development of new therapies should be carried out taking into account this suggestion. In the present investigation we evaluated the efficacy of a treatment with liposomes multifunctionalized for crossing the blood-brain barrier and targeting Aß, carried out on young APP/PS1 Tg mice, taken as a model of pre-symptomatic disease stage. Liposomes were administered once a week to Tg mice for 7months, starting at the age of 5months and up to the age of 12 when they display AD-like cognitive and brain biochemical/anatomical features. The treatment prevented the onset of the long-term memory impairment and slowed down the deposition of brain Aß; at anatomical level, prevented both ventricle enlargement and entorhinal cortex thickness reduction, otherwise occurring in untreated mice. Strikingly, these effects were maintained 3months after treatment discontinuation. An increase of Aß levels in the liver was detected at the end of the treatment, then followed also by reduction of brain Amyloid Precursor Protein and increase of Aß-degrading enzymes. These results suggest that the treatment promotes brain Aß clearance by a peripheral 'sink' effect and ultimately affects Aß turnover in the brain. Worth of note, the treatment was apparently not toxic for all the organs analyzed, in particular for brain, as suggested by the lower brain TNF-α and MDA levels, and by higher level of SOD activity in treated mice. Together, these findings promote a very early treatment with multi-functional liposomes as a well-tolerated nanomedicine-based approach, potentially suitable for a disease-modifying therapy of AD, able to delay or prevent relevant features of the disease.

Dipalmitoylphosphatidic acid inhibits tumor growth in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis, accounting for approximately 12-24% of breast cancer cases. Accumulating evidence has indicated that there is no effective targeted therapy available for TNBC. Dipalmitoylphosphatidic acid (DPPA) is a bioactive phospholipid. However, the function of DPPA in the growth of TNBC has not yet been studied. In this study, we employed TNBC cells and a subcutaneous tumor model to elucidate the possible effect of DPPA on tumor growth in TNBC. We showed that DPPA significantly inhibited tumor growth in the mouse subcutaneous tumor model and suppressed cell proliferation and angiogenesis in TNBC tumor tissues. This inhibition was mediated partly by suppressing the expression of cyclin B1 (CCNB1), which directly promoted the accumulation of cells in the G2 phase and arrested cell cycle progression in human TNBC. In addition, the inhibition of tumor growth by DPPA may also be mediated by the suppression of tumor angiogenesis in TNBC. This work provides initial evidence that DPPA might be vital as an anti-tumor drug to treat TNBC.

Nanoliposomes protect against human arteriole endothelial dysfunction induced by ß-amyloid peptide.

We tested whether nanoliposomes containing phosphatidylcholine, cholesterol and phosphatidic acid (NLPA) prevent ß-amyloid 1-42 (Aß42) fibrillation and Aß42-induced human arteriole endothelial dysfunction. NLPA abolished Aß42 fibril formation (thioflavin-T fluorescence/electron microscopy). In ex-vivo human adipose and leptomeningeal arterioles, Aß42 impaired dilator response to acetylcholine that was reversed by NLPA; this protection was abolished by L-NG-nitroarginine methyl ester. Aß42 reduced human umbilical vein endothelial cell NO production that was restored by NLPA. Nanoliposomes prevented Aß42 amyloid formation, reversed Aß42-induced human microvascular endothelial dysfunction and may be useful in Alzheimer's disease.

Cyclic phosphatidic acid induces G0/G1 arrest, inhibits AKT phosphorylation, and downregulates cyclin D1 expression in colorectal cancer cells.

Lysophosphatidic acid (LPA) and its analogs are well-known mitogens for various cell types. Many reports have confirmed that several types of cancer cell produce LPA to promote survival, growth and tumorigenesis. This indicates that the interface between the LPA signaling pathway and the cell cycle signaling system is critical to the control of cancer cell proliferation. However, our previous study indicated that cyclic phosphatidic acid (cPA), which is structurally similar to LPA, inhibits the proliferation and migration of colon cancer cells. It has been reported that cPA shows several biological activities not shown by LPA. However, understanding of the detailed molecular and cellular mechanism underlying the regulation of the cell cycle by cPA is still in its infancy. In this study, we investigated the effect of cPA treatment on human DLD-1 colon cancer cells by analyzing cell cycle dynamics, gene expression, and AKT phosphorylation. Our findings indicate that cPA inhibits cell cycle progression in DLD-1 colon cancer cells via the downregulation of cyclin D1 and the inhibition of AKT phosphorylation.

Cyclic phosphatidic acid treatment suppress cuprizone-induced demyelination and motor dysfunction in mice.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.

Cyclic phosphatidic acid relieves osteoarthritis symptoms.

BACKGROUND: Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Natural cPA and its chemically stabilized cPA derivative, 2-carba-cPA (2ccPA), inhibit chronic and acute inflammation, and 2ccPA attenuates neuropathic pain. Osteoarthritis (OA) is a degenerative disease frequently associated with symptoms such as inflammation and joint pain. Because 2ccPA has obvious antinociceptive activity, we hypothesized that 2ccPA might relieve the pain caused by OA. We aimed to characterize the effects of 2ccPA on the pathogenesis of OA induced by total meniscectomy in the rabbit knee joint. RESULTS: Intra-articular injection of 2ccPA (twice a week for 42 days) significantly reduced pain and articular swelling. Histopathology showed that 2ccPA suppressed cartilage degeneration in OA. We also examined the effects of 2ccPA on the inflammatory and catabolic responses of human OA synoviocytes and chondrosarcoma SW1353 cells in vitro. 2ccPA stimulated synthesis of hyaluronic acid and suppressed production of the metalloproteinases MMP-1, -3, and -13. However, it had no effect on the production of interleukin (IL)-6, an inflammatory cytokine. The suppressive effect of 2ccPA on MMP-1 and -3 production in synoviocytes and on MMP-13 production in SW1353 cells was not mediated by the lysophosphatidic acid receptor, LPA1 receptor (LPA1R). CONCLUSIONS: Our results suggest that 2ccPA significantly reduces the pain response to OA by inducing hyaluronic acid production and suppressing MMP-1, -3, and -13 production in synoviocytes and chondrocytes.

Orally administered phosphatidic acids and lysophosphatidic acids ameliorate aspirin-induced stomach mucosal injury in mice.

BACKGROUND: Recent investigations revealed that lysophosphatidic acid (LPA), a phospholipid with a growth factor-like activity, plays an important role in the integrity of the gastrointestinal tract epithelium. AIM: This paper attempts to clarify the effect of orally administered phosphatidic acid (PA) and LPA on aspirin-induced gastric lesions in mice. MATERIALS AND METHODS: Phospholipids, a free fatty acid, a diacylglycerol and a triglyceride at 1 mM (5.7 µmol/kg body weight) or 0.1 mM were orally administered to mice 0.5 h before oral administration of aspirin (1.7 mmol/kg). The total length of lesions formed on the stomach wall was measured as a lesion index. Formation of LPA from PA in the mouse stomach was examined by in vitro (in stomach lavage fluid), ex vivo (in an isolated stomach) and in vivo (in the stomach of a living mouse) examinations of phospholipase activity. RESULTS: Palmitic acid, dioleoyl-glycerol, olive oil and lysophosphatidylcholine did not affect the aspirin-induced lesions. In contrast, phosphatidylcholine (1 mM), LPA (1 mM) and PA (0.1, 1 mM) significantly reduced the lesion index. Evidence for formation of LPA from PA in the stomach by gastric phospholipase A2 was obtained by in vitro, ex vivo and in vivo experiments. An LPA-specific receptor, LPA2, was found to be localized on the gastric surface-lining cells of mice. CONCLUSION: Pretreatment with PA-rich diets may prevent nonsteroidal anti-inflammatory drug-induced stomach ulcers.

Effects of cyclic phosphatidic acid on delayed neuronal death following transient ischemia in rat hippocampal CA1.

Cyclic phosphatidic acid (cPA) is a lipid mediator that elicits a neurotrophin-like action in embryonic hippocampal neurons in vitro. In this study, we investigated the effects of cPA and 2-O-carba-oleoyl-cPA (2ccPA), a metabolically stabilized cPA derivative, on ischemia-induced delayed neuronal death in the rat hippocampal CA1 region. Transient occlusion for 8 min of bilateral carotid arteries besides permanent ligation of bilateral vertebral arteries was performed and morphological changes of the neurons were examined histologically 5 days after occlusion. cPA or 2ccPA was continuously administered for 5 days by means of an osmotic pump that was implanted subcutaneously before occlusion. Five days after occlusion, delayed neuronal death occurred in approximately 85% of the CA1 hippocampal neurons in the 0.2-2% bovine serum albumin vehicle control group. However, administration of cPA significantly increased the number of undamaged neurons in a dose-dependent manner. At the most effective concentration (18 µg/kg/5d), the number of undamaged neurons was increased to 4 times of that in the vehicle control group. 2ccPA also showed a neuroprotective effect, but it was less potent than that of natural cPA. These results indicate that systemic administration of both cPA and 2ccPA can protect neurons from ischemia-induced delayed neuronal death in the hippocampus.

Anti-neovascular therapy by liposomal DPP-CNDAC targeted to angiogenic vessels.

We previously reported that liposomalized 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (DPP-CNDAC), a hydrophobized derivative of the novel antitumor nucleoside CNDAC, is quite useful for cancer therapy. On the other hand, for anti-neovascular therapy, we recently isolated peptides homing to angiogenic vessels from a phage-displayed random peptide library, and observed that peptide-modified liposomal adriamycin strongly suppressed tumor growth, perhaps through damaging angiogenic endothelial cells. In the present study, we modified DPP-CNDAC-liposomes with one of the angiogenic homing peptides, APRPG, and examined their antitumor activity. Three doses of APRPG-modified DPP-CNDAC-liposomes (15 mg/kg as CNDAC) strongly inhibited tumor growth compared with the same number of doses of unmodified DPP-CNDAC-liposomes. The life span was increased 31.8%, with one completely cured mouse out of the six mice treated. Since the accumulation of liposomes in the tumor tissue was not so much different between APRPG-liposomes and non-modified liposomes, the enhanced therapeutic efficacy may be explained as the alteration of targets, i.e. APRPG-modified DPP-CNDAC-liposomes caused tumor growth suppression through damage of angiogenic endothelial cells. Anti-neovascular therapy promises no drug resistance, and should be effective against essentially any kind of solid tumor; and thus the present results demonstrate another benefit of the therapy, namely, high efficacy of cancer treatment.

Therapeutic effect of reticuloendothelial system (RES)-avoiding liposomes containing a phospholipid analogue of 5-fluorouracil, dipalmitoylphosphatidylfluorouridine, in Meth A sarcoma-bearing mice.

Reticuloendothelial system (RES)-avoiding liposomes are known to accumulate in tumor tissues due to passive targeting. Dipalmitoylphosphatidylfluorouridine (DPPF), a potent antitumor agent readily incorporated into the lipid bilayer, was embedded in RES-avoiding liposomes modified with a uronic acid derivative, palmityl-D-glucuronide (PGlcUA). The therapeutic effect of DPPF in PGlcUA-liposomes was examined in tumor-bearing mice. Free or liposomal DPPF was injected intravenously into BALB/c mice bearing subcutaneously implanted Meth A sarcomas. The RES-avoiding liposomal formulation using PGlcUA was effective in reducing tumors, and prolonging survival time compared with free DPPF and also DPPF in conventional liposomes. Therefore, PGlcUA-liposomes might be of practical use as drug carriers for anticancer agents, especially their derivatives for embedding in liposomal membranes.

Isolation of experimental anti-AIDS glycerophospholipids by micro-preparative reversed-phase high-performance liquid chromatography.

The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.

Evaluation of incorporation characteristics of mitoxantrone into unilamellar liposomes and analysis of their pharmacokinetic properties, acute toxicity, and antitumor efficacy.

Mitoxantrone (MTO) was incorporated into small unilamellar liposomes by formation of a complex between the anticancer drug and negatively charged lipids. The complex was formed at a 2:1 molar ratio between the lipids and MTO, with phosphatidic acid (PA) being the strongest complex-forming lipid. Weaker complexes and lower incorporation rates of MTO resulted when liposomes containing dicetylphosphate, phosphatidyl inositol, phosphatidyl serine, phosphatidyl glycerol, oleic acid, and tridecylphosphate were used. Thus, all further experiments were performed with PA-MTO liposomes that contained 0.1-3 mg MTO/ml and had mean vesicle sizes of 40-150 nm, depending on the drug concentration and the method of liposome preparation. In vitro incubations of free and liposomal MTO with human plasma showed that the drug is slowly transferred from the liposome membranes to the plasma proteins. For liposomal MTO a transfer rate of 48% was determined, whereas 75.8% of free MTO was bound to the plasma proteins. The organ distribution of the two preparations in mice showed that higher and longer-lasting concentrations of liposomal MTO were found in the liver and spleen. The terminal elimination halflives in the liver were 77 h for liposomal MTO and 14.4 h for free MTO. In the blood, slightly higher concentrations were detected for liposomal MTO, which also had slower biphasic elimination kinetics as compared with the free drug. Drug distribution in the heart was not significantly different from that in the kidneys. The LD25 of PA-MTO liposomes in mice was 19.6 mg/kg and that of free MTO was 7.7 mg/kg. The antitumor effects of PA-MTO liposomes were evaluated in murine L1210 leukemia, in various xenografted human tumors, and in methylnitrosourea-induced rat mammary carcinoma. Generally, the liposomal application form was more effective and less toxic than the free drug. The cytostatic effects were dependent on the tumor model, the application schedule, and the drug concentration. At doses that were toxic when free MTO was used, the liposomal preparation produced strong antitumor effects in some cases. In summary, the incorporation of MTO into liposomes changes the drug's plasma-binding properties, alters its organ distribution, reduces its acute toxicity, and increases its cytostatic efficiency in various tumor models. The liposomal PA-MTO complex represents a new application form of MTO that has advantageous properties.