Encapsulation of an anticancer drug Isatin inside a host nano-vehicle SWCNT: a molecular dynamics simulation.

The use of carbon nanotubes as anticancer drug delivery cargo systems is a promising modality as they are able to perforate cellular membranes and transport the carried therapeutic molecules into the cellular components. Our work describes the encapsulation process of a common anticancer drug, Isatin (1H-indole-2,3-dione) as a guest molecule, in a capped single-walled carbon nanotube (SWCNT) host with chirality of (10,10). The encapsulation process was modelled, considering an aqueous solution, by a molecular dynamics (MD) simulation under a canonical NVT ensemble. The interactions between the atoms of Isatin were obtained from the DREIDING force filed. The storage capacity of the capped SWCNT host was evaluated to quantify its capacity to host multiple Isatin molecules. Our results show that the Isatin can be readily trapped inside the volume cavity of the capped SWCNT and it remained stable, as featured by a reduction in the van der Waals forces between Isatin guest and the SWCNT host (at approximately - 30 kcal mol-1) at the end of the MD simulation (15 ns). Moreover, the free energy of encapsulation was found to be - 34 kcal mol-1 suggesting that the Isatin insertion procedure into the SWCNT occurred spontaneously. As calculated, a capped SWCNT (10,10) with a length of 30 Å, was able to host eleven (11) molecules of Isatin, that all remained steadily encapsulated inside the SWCNT volume cavity, showing a potential for the use of carbon nanotubes as drug delivery cargo systems.

A Neuroprotective Dose of Isatin Causes Multilevel Changes Involving the Brain Proteome: Prospects for Further Research.

Isatin (indole-2,3-dione) is an endogenous regulator, exhibiting a wide range of biological and pharmacological activities. At doses of 100 mg/kg and above, isatin is neuroprotective in different experimental models of neurodegeneration. Good evidence exists that its effects are realized via interaction with numerous isatin-binding proteins identified in the brain and peripheral tissues studied. In this study, we investigated the effect of a single dose administration of isatin to mice (100 mg/kg, 24 h) on differentially expressed proteins and a profile of the isatin-binding proteins in brain hemispheres. Isatin administration to mice caused downregulation of 31 proteins. However, these changes cannot be attributed to altered expression of corresponding genes. Although at this time point isatin influenced the expression of more than 850 genes in brain hemispheres (including 433 upregulated and 418 downregulated genes), none of them could account for the changes in the differentially expressed proteins. Comparative proteomic analysis of brain isatin-binding proteins of control and isatin-treated mice revealed representative groups of proteins sensitive to isatin administration. Control-specific proteins (n = 55) represent specific targets that interact directly with isatin. Appearance of brain isatin-binding proteins specific to isatin-treated mice (n = 94) may be attributed to the formation of new clusters of protein-protein interactions and/or novel binding sites induced by a high concentration of this regulator (ligand-induced binding sites). Thus, isatin administration produces multiple effects in the brain, which include changes in gene expression and also profiles of isatin-binding proteins and their interactomes. Further studies are needed for deeper insight into the mechanisms of the multilevel changes in the brain proteome induced by isatin. In the context of the neuroprotective action, these changes may be aimed at interruption of pathological links that begin to form after initiation of pathological processes.

Pre-clinical evidence of safety and protective effect of isatin and oxime derivatives against malathion-induced toxicity.

The inhibition of acetylcholinesterase (AChE) is a common outcome caused by organophosphorus (OPs) intoxication. Although inconsistent, the standard treatment consists of a muscarinic receptor antagonist (atropine) and AChE-reactivating molecules such as oximes. This study proposes to test unpublished compounds which contain the moieties of isatin and/or oxime have protective effects against the toxicity induced by malathion in two animal models: Artemia salina and Rattus norvegicus (Wistar rats). The lethality was assessed in A salina, and the calculated LD50 to (3Z)-5-chloro-3-(hydroxyimino) indolin-2-one oxime (Cℓ-HIN) and 2-(5-chloro-2-oxoindolin-3-ylidene)-hydrazinecarbothioamide (Cℓ-OXHS) was higher than 1000 µM while to 3-(phenylhydrazono) butan-2-one oxime (PHBO) was 38 µM. Our screening showed that Cℓ-HIN seems to be the most promising molecule, with low toxicity to A salina, protection against mortality (with or without atropine) and AChE inhibition induced by malathion. Similarly, the oral administration of 300 mg/kg of Cℓ-HIN induced low or no toxicity in rats. The plasma butyrylcholinesterase (BChE) and cortical AChE activities were reactivated by Cℓ-HIN (50 mg/kg, p.o.) in rats exposed to malathion (250 mg/kg, i.p). No difference was observed in paraoxonase-1 (PON-1) activity among groups treated. In conclusion, Cℓ-HIN restored the cholinesterase activities inhibited by malathion in A salina and rats with low toxicity in both. Thus, the data provide evidence that Cℓ-HIN, a compound that combines isatin and oxime functional groups, is safe and has important properties to reactivate the cholinesterases inhibited by malathion. In addition, we demonstrate the importance of a preliminary assessment in an alternative model in order to reduce the use of mammalians in drug discovery.

Antiproliferative activity and possible mechanism of action of certain 5-methoxyindole tethered C-5 functionalized isatins.

BACKGROUND: Cancer is one of the most dreaded human diseases, that has become an ever-increasing health problem and is a prime cause of death globally. The potential antiproliferative activity of certain indole-isatin molecular hybrids 5a-w was evaluated in vitro against three human cancer cell lines. METHODS: Standard protocols were adopted to examine the antiproliferative potential and mechanisms of compounds 5a-w. Western blot analysis was carried out on compound 5o. RESULTS: Compounds 5a-w demonstrated in vitro antiproliferative activity in the range of 22.6-97.8%, with compounds 5o and 5w being the most active antiproliferative compounds   with IC50 values of 1.69 and 1.91 µM, which is fivefold and fourfold more potent than sunitinib (IC50=8.11 µM), respectively. Compound 5o was selected for in-depth pharmacological testing to understand its possible mechanism of antiproliferative activity. It caused a lengthening of the G1 phase and a reduction in the S and G2/M phases of the cell cycle and had an IC50 value of 10.4 µM with the resistant NCI-H69AR cancer cell line. Moreover, compound 5o significantly decreased the amount of phosphorylated Rb protein in a dose-dependent fashion, which was confirmed via Western blot analysis. CONCLUSION: The current investigation highlighted the potential antiproliferative activity of compounds 5a-w as well as the antiproliferative profile of compound 5o. These compounds can be harnessed as new lead antiproliferatives in the preclinical studies of cancer chemotherapy.

[The effect of deprenyl and isatin administration to mice on the proteomic profile of liver isatin-binding proteins]. / Vliianiia vvedeniia deprenila i izatina mysham na profil' izatin-sviazyvaiushchikh belkov pecheni.

Isatin (indol-2,3-dione) is an endogenous indole found in the brain, peripheral tissues and biological body fluids of humans and animals. Its wide spectrum of biological activity is realized via interaction with numerous isatin-binding proteins; these include proteins playing an important role in the development of neurodegenerative pathology. In the context of the neuroprotective effect, the effect of isatin is comparable to the effects of deprenyl, a pharmacological agent used for treatment of Parkinson's disease. In this study, the effects of the course of deprenyl (1 mg/kg) and isatin (20 mg/kg) administration for 21 days on the profile of the isatin-binding proteins of the liver of mice have been investigated. Proteomic profiling of liver isatin-binding proteins of control mice by means of 5-aminocaproylisatin as an affinity ligand resulted in identification of 105 proteins. Treatment of animals with a low dose of isatin slightly decreased (up to 91), while injections of deprenyl slightly increased (up to 120) the total number of isatin-binding proteins. 75 proteins were common for all three groups; they represented from 62.5% (in deprenyl treated mice) and 71% (in control mice), to 82% (isatin treated mice) of the total number of identified liver isatin-binding proteins. Proteomic analysis of the isatin-binding proteins of mice treated with isatin (20 mg/kg) or deprenyl (1 mg/kg) for 21 days revealed a representative group of proteins (n=30) that were sensitive to the administration of these substances. Taking into account the previously obtained results, it is reasonable to suggest that the change in the profile of isatin-binding proteins may be attributed to accumulation of isatin and deprenyl in the liver and interaction with target proteins prevents their subsequent binding to the affinity sorbent. In this context, the identified isatin-binding liver proteins of control animals that do not bind to the affinity sorbent (immobilized isatin analogue) after treatment of animals with either deprenyl or isatin appear to be specific targets directly interacting with isatin in vivo.

Oestrogen receptor-mediated liposomal drug delivery for treating melanoma.

Function of steroid hormone oestrogen that transactivates oestrogen receptor (ER) is expressed in multiple organs. Except for malignancies of gynaecological organs, ER remains largely unutilised as a target to treat cancers of ER-expressing brain, prostate, skin etc. We have previously developed oestrogen targeting cationic lipid molecule (ES-C10), which showed targeted killing of ER + breast and skin cancer cells. In this study, we explored the targeting ability of ES-C10 as a ligand as well as its additive killing effect (if any), when incorporated in two different liposomes (DCME and DCDE), carrying two anticancer molecules MCIS3 and Docetaxel™, respectively. DCME and DCDE exhibited higher cytotoxicity in ER + cancer cells than in ER - cancer or in non-cancer cells. Both liposomes induced ER-mediated cytotoxicity and caspase 3-induced apoptosis in ER + melanoma cells. Further, decreased levels of pAkt, and increased levels of PTEN and p53 were also observed. Both the targeted liposomes were least haemolytic. These selectively delivered drug-cargoes to tumour mass over other vital organs and induced better anti-tumour effect, which led to increased survivability than their respective controls. In conclusion, we demonstrated the development of two independent liposomal drug-delivery systems associated with an anticancer, oestrogen-structure based ligand for efficient, ER-mediated anti-melanoma effect.

Targeting urokinase and the transferrin receptor with novel, anti-mitotic N-alkylisatin cytotoxin conjugates causes selective cancer cell death and reduces tumor growth.

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo.

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.

Isatin down-regulates expression of atrial natriuretic peptide receptor A and inhibits airway inflammation in a mouse model of allergic asthma.

Isatin, an endogenous indole compound, prevents atrial natriuretic peptide (ANP) from signaling through its cell-surface receptor, NPRA. Allergic airway inflammation has been linked to natriuretic peptide signaling and blocking this signaling axis in the lung prevents allergen-induced pathology. In this study we encapsulated isatin in chitosan nanoparticles and tested them in a mouse model of allergic asthma by intranasal delivery to the lung. Isatin nanocapsules reduced lung pathology by blocking ANP signaling, but surprisingly also by reducing the expression of NPRA. Ovalbumin-allergic mice were treated intranasally with isatin-containing chitosan nanocapsules either before or after allergen challenge, and lung function, cytokine levels, histopathology and cellular infiltration were determined. ANP activity was quantitated by measuring changes in intracellular cyclic GMP and changes in NPRA levels were determined. For comparison with isatin's effects, the expression of the receptor was inhibited with small interfering RNA against NPRA mRNA. Isatin nanocapsules administered locally to the lung reduced cGMP production and NPRA expression and protected allergic mice from airway hyperreactivity and lung inflammation when given either before or after allergen challenge. Leukocyte infiltration was reduced and lung cytokine profiles showed a repolarization from a Th2-like to a Th1-like phenotype. Isatin nanocapsules administered locally to the lung inhibit NPRA signaling but also are capable of lowering the expression of NPRA, thus effectively reducing inflammation in a mouse model of allergic asthma. Pharmacological intervention to reduce NPRA activity through the inflammatory natriuretic peptide axis in the lung may be a useful adjunct therapy for treating lung disease.

The effect of short-term administration of (-)-deprenyl and isatin on the expressions of some genes in the mouse brain cortex.

BACKGROUND: Isatin (indoledione 2,3) is an endogenous indole found in the mammalian brain, peripheral tissues, and body fluids. It exhibits many neurophysiological and neuropharmacological effects. It shares some common molecular targets with (-)-deprenyl, a neuroprotective pharmacological drug. Some isatin effects imply a possible influence of gene expression; however, no isatin-responsive genes have yet been identified. MATERIAL/METHODS: In this study the effects of a three-week administration of isatin (20 mg/kg) or (-)-deprenyl (1 mg/kg) on the expressions of several putative isatin/deprenyl-responsive genes in the mouse cortex were compared using real-time PCR. RESULTS: Both treatments caused similarly significant decreases in superoxide dismutase (SOD) mRNA. Treatment of mice with either drug decreased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, although only in the deprenyl-treated mice was this significant (p<0.01). No significant changes were found in cortex mRNA content of monoamine oxidase A or monoamine oxidase B. CONCLUSIONS: The results suggest that isatin and (-)-deprenyl have some common target genes and this supports the idea that isatin may be an endogenous partial functional agonist of (-)-deprenyl. Since GAPDH mRNA expression is sensitive to the pharmacological treatments, these results also question the applicability of GAPDH as a reference gene in gene expression studies.

In vivo effects of isatin on rat platelet eicosanoids.

To establish the possible influence of isatin (2,3-dioxo-indole) on the activity of platelets, the effects of isatin on platelet eicosanoid synthesis were studied in rats. Different doses (12.5-50 mg/kg) of isatin were injected intraperitoneally (i.p.) and the effects on the arachidonate cascade of isolated platelets were investigated. Cells were labeled with [(14)C]arachidonic acid, then the eicosanoids were separated with overpressure thin-layer chromatography and were quantitatively determined with a liquid scintillation analyzer. The lipoxygenase pathway was significantly inhibited by isatin (50 mg/kg) treatment and also the overall activity of the arachidonate cascade was diminished; however, the cyclooxygenase system was significantly stimulated. A 50-mg/kg i.p. dose of isatin significantly increased the production of vasoconstrictor cyclooxygenase metabolites. Among the vasodilator cyclooxygenase products, the synthesis of PGE2 and PGD2 were significantly decreased while that of 12-hydroxyheptadecatrienoic acid (HHT) increased upon isatin (50 mg/kg) administration. Our results provide further evidence on the peripheral actions of isatin and suggest that this endogenous indole may induce significant changes in the production of blood platelet arachidonic acid metabolites, which are important regulatory substances, thus isatin may potentially affect an even broader range of functions than was previously assumed.

Synthesis, antitubercular activity and pharmacokinetic studies of some Schiff bases derived from 1-alkylisatin and isonicotinic acid hydrazide (INH).

N'-(1-alkyl-2,3-dihydro-2-oxo-1H-3-indolyliden)-4-pyridinecarboxylic acid hydrazide derivatives, 3(a-g), were synthesized in a trial to overcome the resistance developed with the therapeutic uses of isoniazid (INH). The lipophilicity of the synthesized derivatives supersedes that of the INH as expressed by Clog p values. The synthesized compounds and INH were tested against bovin, human sensitive and human resist strains of Mycobacterium tuberculosis. Compounds 3a, 3d, 3f and 3g with 1-unsubstituted, 1-propyl, 1-propynyl and 1-benzyl groups respectively exhibited equipotent growth inhibitory activity (MIC 10 micromol) against the tested strains as compared with INH however the later has no activity against human resist strain. Pharmacokinetic study revealed that the rate and extent of absorption of the tested derivatives (3d and 3f) significantly higher than that of INH (p < 0.05). The relative bioavailabilities (F(R)%) were 183.15 and 443.25 for 3f and 3d respectively as compared to INH. These results preliminary indicate the possible use of the prepared derivatives for treatment of tuberculosis infections in order to overcome the resistance developed with INH.

The effects of isatin (indole-2, 3-dione) on pituitary adenylate cyclase-activating polypeptide-induced hyperthermia in rats.

BACKGROUND: Previous studies have demonstrated that centrally administered natriuretic peptides and pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) have hyperthermic properties. Isatin (indole-2, 3-dione) is an endogenous indole that has previously been found to inhibit hyperthermic effects of natriuretic peptides. In this study the aim was to investigate the effects of isatin on thermoregulatory actions of PACAP-38, in rats. RESULTS: One microg intracerebroventricular (icv.) injection of PACAP-38 had hyperthermic effect in male, Wistar rats, with an onset of the effect at 2 h and a decline by the 6th h after administration. Intraperitoneal (ip.) injection of different doses of isatin (25-50 mg/kg) significantly decreased the hyperthermic effect of 1 microg PACAP-38 (icv.), whereas 12.5 mg/kg isatin (ip.) had no inhibiting effect. Isatin alone did not modify the body temperature of the animals. CONCLUSION: The mechanisms that participate in the mediation of the PACAP-38-induced hyperthermia may be modified by isatin. The capability of isatin to antagonize the hyperthermia induced by all members of the natriuretic peptide family and by PACAP-38 makes it unlikely to be acting directly on receptors for natriuretic peptides or on those for PACAP in these hyperthermic processes.

Synthesis of isatin semicarbazones as novel anticonvulsants--role of hydrogen bonding.

PURPOSE: A series of substituted isatin semicarbazones and related bioisosteric hydrazones were designed and synthesised to meet the structural requirements essential for anticonvulsant properties. METHODS: The structures of all synthesised compounds were confirmed by means of infrared, proton magnetic resonance spectroscopy and by elemental analyses. All compounds were evaluated for their anticonvulsant activity by maximal electroshock (MES), subcutaneous metrazol (ScMet) and subcutaneous strychnine (ScSty) induced seizure methods and their neurotoxic effects were determined by rotorod test. RESULTS: A number of isatin semicarbazones exhibited significant protection after intraperitoneal administration at the dose of 100 and 300mg/kg. Some of them showed good anticonvulsant activity in MES test in rats after per oral administration at the dose of 30mg/kg. The bioisosteric hydrazone derivatives were inactive in all tests. Compound 6-chloroisatin-3- (4-bromophenyl)-semicarbazone has emerged as the most active analogue of the series showing good activity in all the three tests and was more active than phenytoin and valproic acid. CONCLUSIONS: The results evidenced the importance of hydrogen bonding and suggested a new pharmacophore model with four binding sites essential for anticonvulsant activity.

Isatin (Indole-2, 3-dione) inhibits natriuretic peptide-induced hyperthermia in rats.

The effects of an endogenous indole, isatin (indole-2, 3-dione), on the hyperthermia induced by atrial natriuretic peptide (ANP-28), brain natriuretic peptide (BNP-32), and C-type natriuretic peptide (CNP-22) were investigated in rats. Intracerebroventricular administration of each peptide in a dose of 1 microg caused elevations in colon temperature 30 and 60 min after injection. An intraperitoneal (i.p.) injection of isatin (50 mg/kg) abolished the natriuretic peptide-induced hyperthermia. These data reinforce the possible involvement of natriuretic peptides in thermoregulatory processes in the central nervous system, and suggest that isatin might counteract their hyperthermic effect in vivo.

Dose-related proconvulsant and anticonvulsant activity of isatin, a putative biological factor, in rats.

Isatin (indole-2, 3-dione) is an endogenous compound with anxiogenic properties, which occur within a narrow dose range (15-20 mg/kg, i.p.). Dose increment beyond 50 mg/kg, i.p. leads to the loss of anxiogenesis. Since a link has been postulated between anxiogenic and convulsant activity, the effect of a range of doses of isatin (20-80 mg/kg, i.p.) was investigated on subconvulsant and convulsant doses of two seizure-inducing agents, namely, pentylenetetrazole (PTZ) and 3-mercapto-propionic acid (3MPA) in rats. Isatin was found to induce a dose-related effect on PTZ and 3MPA convulsions. The lower dose (20 mg/kg, i.p.) potentiated PTZ and 3MPA convulsions, a median dose (40 mg/kg, i.p.) had insignificant effect, whereas higher doses (60 and 80 mg/kg, i.p.) of isatin exhibited significant anticonvulsant effect against both PTZ and 3MPA induced clonic convulsions. The investigation, thus, supports the contention that anxiogenic agents increase the susceptibility to chemical seizures. The proconvulsant effect of isatin, may be due to its inhibitory effect on central atrial natriuretic peptide receptors and stimulation of 5-hydroxytryptamine3 (5-HT3) rather than its monoamine oxidase (MAO) B inhibitory action. The anticonvulsant effect on higher doses of isatin, on the contrary, may be induced by its metabolites, including 5-hydroxyisatin.

Determination of isatin, an endogenous monoamine oxidase inhibitor, in urine and tissues of rats by HPLC.

1. We have previously identified isatin as one of the endogenous monoamine oxidase (MAO) inhibitors in the urine and the brain of stroke-prone spontaneously hypertensive rats (SHRSP), using gas chromatography-mass spectrometry (GC-MS). 2. In this study, we attempted to develop a convenient assay to determine isatin using high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The standard curve for authentic isatin was linear at a range from 2 to 20 nmol per ml. The coefficient of variance was within 3% for both intra-assay and inter-assay. The sensitivity was 20 pmol per 10 microl of urine sample. 3. Isatin concentration correlated significantly and positively with endogenous MAO activity (tribulin-like activity) in both urine (r=0.924, P<0.001) and kidney extracts (r=0.862, P<0.01). There was a significant difference in urinary isatin between Wistar Kyoto rats (WKY) and SHRSP. Oral administration of isatin increased urinary isatin concentration and systolic blood pressure in WKY. 4. Determination of isatin using HPLC-UV may be useful for elucidating role of isatin in various conditions of stress and disease.

Antiviral activity of certain isatinisothiosemicarbazones against Mengo and vaccinia virus infections in mice.

Among eleven isatinisothiosemicarbazones (IITS) examined only 1-ethylisatin-S-n-butylisothiosemicarbazone (ZIMET 189/69) proved to be significantly effective against lethal Mengo and neurovaccinia virus-induced encephalitis in mice when administered subcuaneously (s.c.) in doses of 1 mmole/kg body weight per day. With peroral (p.o.) administration all the drugs failed to prevent mortality in Mengo virus-infected mice. Against this virus ZIMET 189/69 was effective over a concentration range from 0.6-1.4 mmoles/kg per day producing a plateau effect at doses greater than 1 mmole/kg. With Mengo virus maximum protective response was seen in intraperitoneally (i.p.) or intranasally (i.n.) infected mice when treatment was begun at the time of virus inoculation and continued, once daily for a period of at least four consecutive days. Delayed initiation of treatment failed to protect Mengo virus-infected mice but was effective in vaccinia virus-infected mice.