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Three organometallic complexes derived from pentamidine were evaluated for their trypanocidal effect on in vivo Trypanosoma brucei brucei models in comparison to pentamidine isethionate as reference compound. On the T. b.brucei mouse model, the most active compound was cis-platinum-pentamidine bromide. This compound was active when subcutaneously administered at the single dose of 1.5 micromol/kg and its chemotherapeutic index was 200 whereas pentamidine isethionate was active at 6 micromol/kg with a chemotherapeutic index of 13, when administered in the same conditions. Cis-platinum-pentamidine bromide was active at 1 mg/kg (1.44 mmoles/kg), in a single dose by subcutaneous route against the early stage of the T. b.brucei Antat 1-9 sheep model. Platinum kinetics in serum showed a Cmax of 0.2 mg/l reached 80 h after the treatment at this dose. Cis-platinum-pentamidine bromide, cis-platinum-pentamidine seleniocyanate, and cis-platinum-pentamidine thiocyanate were distributed in the deep compartment according to a monocompartmental model. In all cases, platinum was eliminated from the serum 700 hours post-treatment. All data obtained from these models show activity on the early stage of the disease and justify further investigations on the late stage of the disease.

Ir-(COD)-pentamidine tetraphenylborate which has previously been studied on promastigote forms of Leishmania, was investigated for its antileishmanial properties compared with pentamidine used as reference compound. In vitro, the iridium complex had the same IC50 value on intracellular forms of Leishmania as pentamidine (15 microM). In vivo, the compound could not be injected intravenously due to the DMSO excipient so that the treatments were performed intraperitoneally or subcutaneously. On the L. donovani LV9/Balb/C mouse model, the iridium complex was not toxic after intraperitoneal treatment at 232 mg/kg/day x 5 or 147 mumoles/kg/day x 5, whereas all the mice died within five days when treated at the same dose with pentamidine isethionate. However, only 23% of parasite suppression was observed with the iridium complex. On a L. major MON 74/Balb/C mouse model, susceptible to intravenously administered pentamidine at 6.7 mumoles/kg/day x 5 (54% of parasite suppression), the iridium complex exhibited 32% of parasite suppression after a treatment at 76 mumoles/kg/day x 5 administered subcutaneously. This slight activity is of interest since pentamidine isethionate is not active under these conditions. Transmission electron microscopy of amastigotes from infected and treated mice show aggregation of ribosomal material, distension of the nuclear membrane and kDNA depolymerization. The mechanism of action therefore involves several targets: membranes, ribosomes and kDNA. According to our results, the Iridium complex is a suitable candidate to be encapsulated in drug carriers such as liposomes or nanoparticles.

Iridium (Ir)-(COD)-pentamidine tetraphenylborate (CAS 225-75-4) was selected from a primary screening to be evaluated in vitro on three Leishmania (L.) strains comparatively to pentamidine used as reference compound. The IC50 values obtained from in vitro evaluation on promastigotes of L. major CRE 26, L. donovani DD8 and L. donovani LV9 were 3.9, 23.5, and 3.3 mumol/l for Ir-(COD)-pentamidine tetraphenylborate and 1.6, 7.7, and 3.9 mumol/l for pentamidine isethionate, respectively. Cytotoxicity on mouse peritoneal macrophages led to determine a chemotherapeutic index of 1.7 for Ir-(COD)-pentamidine tetraphenylborate and 4 for pentamidine. Considering L. donovani DD8, the uptake of iridium complex by the promastigotes was shown to be saturable with a Km value of 17.4 mumol/l and Vmax of 1.3 nmol/mg protein/2 h. After 2 and 4 h incubation of treated promastigotes in drug free medium the absence of Ir-complex efflux is in favour of intracellular drug binding. As a matter of fact iridium complex was shown to bind ribosomal subunits in vitro, with no effect on macromolecular biosynthesis.

New organometallic complexes having protozoocidal properties were evaluated for their in vitro antifilarial activity using two models: infective larvae of Molinema dessetae and adult females of Brugia pahangi. The compound most active on the M. dessetae model was Ir(I)-COD-pentamidine tetraphenylborate with an EC50 = 6 +/- 1 microM after 7-day-incubation. In the 2-aminobenzothiazole series, Ruthenium was more potent than Iridium for antifilarial activity. A dithiocarbamate function significantly enhanced the antifilarial activity. The compounds derived from benzimidazole were inactive whatever the metal (Iridium or Rhodium). The other compounds exhibited EC50 ranging from 10 to 31 microM. On adult female Brugia pahangi in vitro, Pt-DDH-N-acetylleucine, Pt-diminazene and Pd-Cl4-piperazine at 20 microM began to kill both microfilariae and the developing embryos within the mothers on day 2. The compounds, except for Pd-Cl4-piperazine, killed the adults after 5 days. Rh-Cl-2-chloropyridine caused obvious slowing of the adults from day 3 onward but did not affect the viability of adults, microfilariae or developing embryos. In vivo antifilarial investigations are necessary to appreciate the real advantage of heavy metal complexes in the experimental treatment of filariasis.

Pentamidine di-(iridium cyclo-octadiene)tetraphenylborate, called Ir-(COD)-pentamidine tetraphenylborate, was selected from a primary screening as a promising trypanocidal compound. The compound was evaluated against three isolates: Trypanosoma brucei brucei CMP, T.b. brucei GVR 35 and T.b. gambiense Feo. On the T.b. brucei GVR 35 murine CNS model, no mouse was cured when the treatment was commenced 21 days post-infection whatever the treatment regimen. Nevertheless, in vitro the compound killed the trypomastigote forms of T. b. gambiense Feo at 0.6 microM. In vivo, the compound cured all mice infected 1 hour previously with T. b. gambiense Feo after a 10 mg/kg (6.3 mumol/kg) treatment subcutaneously administered in a single dose. Moreover, the compound was active at 1 mg/kg (0.6 mumol/kg) in a single dose against the early stage of the T. b. brucei Antat 1-9 sheep model. Serum kinetics data showed that pentamidine di-(iridium cyclo-octadiene) tetraphenylborate was distributed within deep compartment according to a monocompartmental model. The maximum iridium serum concentration was 198 micrograms/l corresponding to 1 mumol/kg of iridium derivative and this value remained stable for 30-50 hours post-treatment. Iridium was completely eliminated from the serum 700 hours post-treatment. all data obtained from these models are in favour of an activity in the early stage of the disease but indicate that the compound could not cross the blood-brain barrier despite its lipophilicity. Although iterative treatments with the compound rapidly induced the selection of iridium derivative refractory populations, the compound could be studied on pentamidine refractory strains.

Stilbamidinium hexachloroiridiate was found trypanocidal in vitro against Trypanosoma brucei brucei IPP at 600 microM after a 1 h incubation period and 30 microM after 24 h. This activity was confirmed in mice with a subcutaneous treatment at 20 mg/kg in a single dose. It was then evaluated on T.b. brucei murine CNS model. At the early stage, a subcutaneous treatment at 2 mg/kg/day x 5 cured 50% mice where-as one single dose at 10 mg/kg was completely inactive. Higher doses failed to cure the mice. Nevertheless, hexachloroiridiate salt of stilbamidine was 3.3 fold less toxic than dihydrochloride salt. Although the compound appeared inactive at the late stage of the murine trypanosomiasis, the difference of toxicity justified its evaluation on the early stage of sheep trypanosomiasis. The compound was trypanocidal at 2 mg/kg in a single dose when administered 8 days after infection. The study of iridium serum kinetic showed that stilbamidinium hexachloroiridiate was distributed rapidly according to a monocompartmental model. Moreover, iridium persisted in serum for a long time. The compound in aqueous suspension with 1% carboxymethylcellulose acted therefore as a controlled release system with a bioavailability allowing its trypanocidal action at the early stage.

New organometallic complexes have been synthesized by association of an active organic molecule with a metallic element such as Pt, Rh, Ir, Pd, Os. Their trypanocidal activity was studied in vitro and in vivo against T. b. brucei. The more active compounds were pentamidine derivatives. The Ir- COD-pentamidine complex, and Iridium (I) cationic and organometallic complex showed and in vitro activity at 60 micrograms/l. Moreover, all infected mice were cured by this compound subcutaneously administered in a single dose at 0.5 mg/kg (0.317 mumol/kg). In the same conditions, pentamidine cured all the mice at 5 mumol/kg. Ir-COD-pentamidine (or P1995) was 16 fold more efficient than pentamidine. Since the chemotherapeutic index of this molecule was 7.5 fold higher than those of pentamidine, P1995 can be considered as a potential trypanocidal drug of the future.

The activities of twenty seven Platinum, Rhodium and Iridium drug complexes were determined against Leishmania donovani amastigotes in mouse peritoneal macrophages in vitro. Eight compounds showed antileishmanial activity of which only three, Rh(III)-mepacrine, Ir(III) pyrrolidine dithiocarbamate and Ir(III) diethyl dithiocarbamate had ED50 values of less than 1 microM. The two Iridium complexes produced, respectively, a 50% and 39% suppression of L. donovani amastigotes in the liver of BALB/c mice following the subcutaneous administration of 200 mg/kg for 5 consecutive days. Ultrastructural studies suggest that the amastigote kinetoplast-mitochondrion complex is the primary site of action of the Ir and Rh complexes.

New rhodium(I) complexes, belonging to the general structure [Rh(CO)2 (L)], where dithiocarbamate and xanthate derivatives, were synthesized and assayed as cytostatic and antitumour agents in vitro against KB cells and in vivo against P388 leukaemia, Ehrlich ascites carcinoma, Sarcoma 180 ascites and ADJ/PC6A solid tumour. Assays against five trypanosoma strains were also performed. Among the new compounds the [Rh(CO)2 (DPA-dtc)] appeared to be active in all biological systems without showing evident nephrotoxicity.

A series of organometallic drugs were tested in a photometric in vitro assay for trypanocidal activity against the three subspecies of the Trypanosoma brucei group. Trypanosoma b. rhodesiense, T. b. gambiense and T. b. brucei displayed a similar sensitivity pattern. The following IC50 values (drug concentration inhibiting decrease of extinction by 50%) could be determined: IrCl6 (IV)-Pentamidine (1.5 ng/ml), IrCl6(IV)-Ro15-0216 (10 micrograms/ml), PtBr6-Metamidium (30 micrograms/ml), PtBr6(IV)-Benznidazole (20 micrograms/ml), PtBr6(IV)-Ro15-0216 (50 micrograms/ml), cis-Pt(II)-Pentamidine (0.1 micrograms/ml), Rh(I)-COD-Pentamidine (5-10 ng/ml), Rh(I)-COD-Benznidazole (10 micrograms/ml), Rh(III)-Ethylxanthate (30 micrograms/ml) and Pt(II)-Pentamidine-cis-Pt(II)(Pentamidine)(Cl)2 (2 ng/ml). The four Pentamidine complexes were the most active compounds and showed activities comparable to that of Pentamidine (IC50: 0.5 to 1 ng/ml). Since the toxicity of the Ir- and Pt-Pentamidine complexes is ten to twenty fold lower (400 to 1000 mg/kg) than that of Pentamidine (50 mg/kg), the complexes might be candidates for investigation as safer trypanocidal drugs.

A model of liver hyperplastic noduligenesis was induced in rats in vivo by long-term administration of thioacetamide (TAM; 100 mg/kg day i.p.). Three doses of 50 mg/kg of an antitumoral rhodium(III) complex were administered at 14, 9 and 5 days before the end of TAM treatment. Blood and liver were obtained from either TAM, Rh(III) complex or TAM plus Rh(III) complex-treated rats in order to determine the interaction of both (tumoral and antitumoral) substances with the biochemical pathways related to glutathione redox cycle, enzyme activities involved in the oxidative stress coupled to the NADPH/NADP pair and enzymes related to the mono-oxygenase P450 system. The results showed that TAM induced an imbalance between the activities of glutathione-coupled enzymes. Glutathione reductase activity increased along with the intoxication, while glutathione peroxidase activity decreased. Alterations in the activity of soluble glutathione peroxidase were parallel to those of catalase. These results, together with decreased activities of enzymes related to cytochrome P450 mono-oxygenase system, NADPH cytochrome P450 reductase and NADH cytochrome b5 reductase, suggest that liver cells are not protected against the peroxidative stress produced by chronic administration of TAM. The Rh(III) complex did not produce significant changes in the parameters assayed when administered alone. When this complex was administered to TAM-treated rats, significant restoration of the following activities was observed: those of NADPH-generating enzymes (glucose-6-phosphate dehydrogenase and malic enzyme), that of glutathione reductase (NADPH-consuming enzyme), NADPH-cytochrome P450 reductase and total catalase. These results, together with others in previous studies, suggest that the altered liver function induced by chronic administration of TAM can be partially restored by this rhodium complex. The mechanisms by which this complex counteracts the TAM-induced changes have not yet been established.

Six new platinum (II)-sulfonamide complexes were examined for their in vitro cytostatic properties as well as in vivo antitumour effect against three experimental murine tumours. The possible antitrypanosomic in vivo activity against T. brucei, T. congolense and T. cruzi infections was also evaluated. The synthesis and chemical characterization of new complexes is reported. Only two sulfadiazine derivatives appeared to be effective mainly against Ehrlich ascites but in a smaller extent than cisplatin. A satisfactory correlation between antitumour and antitrypanosomic activity was found.

Ten new Pt (II) complexes were synthesized and tested as potential antitumor drugs in vitro on KB human tumour cell line, and in vivo against four experimental tumour systems (P388, L1210, ADJ/PC6A and Yoshida sarcoma). The complexes contained two primary amine ligands (cyclopentylamine) with bidentate leaving ligands consisting of nitro-, dinitro- and sulfo-derivatives of phthalic and isophthalic anions. Various complexes showed a good cytostatic effect in vitro with ID50 from 0.29 and 0.99 mcg/ml. The Pt(cpa)2 (5-sulfo-IPA) appeared to be the most effective compound against P388 and L1210 (T/C% 310 and 250 respectively after three 50 mg/Kg i.p. injections) as well as against ADJ/PC6A (ID90 2.8 mg/kg after a single i.p. injection) and Yoshida sarcoma (T/C% 17.5 after a single 50 mg/kg i.p. injection), but the phthalic acid nitro-derivatives were also quite effective. As far as antileukemic effect was concerned, there was a fairly good correlation between the results in vitro and in vivo. Relationships between antitumour activity and pi electronic charge localization on O- atoms of leaving ligands (M.O. Huckel's Calculations) are also discussed.

Six new organometallic derivatives of Rh(I), belonging to the general structure [Rh(CO)2(L)(Cl)], were synthesized and characterized by chemical analysis and IR determinations. The following ligands (L) were employed: 2-aminothiazole, thiazole, 2-amino-6-bromobenzothiazole, 5-chloro-2-methylthiobenzothiazole, 2-bromo-thiazole, and 2-isopropylthiazole. These new complexes were assayed in vitro with KB cells and in vivo with mice bearing established P388 and L1210 leukemias. Assays against S180 and Ehrlich ascitic tumors were also performed. Two complexes displayed antitumor activity against ascitic tumors.

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats.The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration.The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.

Fourteen platinum (Pt) coordination complexes with different ligands, which include both Pt(II) and Pt(IV) complexes, were prepared, characterized and tested for their in vitro cytotoxic effects on KB cells and for their antitumour activity against some tumour systems (L1210 and P388 leukaemia, ADJ/PC6A plasma cell tumour and Yoshida sarcoma). The majority of the ligands were derivatives of aniline or pyridine, but complexes with tranylcypromine, guanethidine and octodrine were also synthetized. Depending on cytotoxicity the Pt-compounds could be divided into 3 groups. The compounds with a high cytotoxicity (ED50 = 0.1-1 microgram/ml) were also active against L1210 and P-388 leukaemia; a correlation between cytotoxicity and antitumour activity was not always observed. In these complexes the oxidation state of the Pt appears to be critical for their activity.

The present report deals with the alterations produced by cis-diamminedichloroplatinum (II) (DDP), and 2 of its analogs: cis-Pt(II)(tranylcypromine)2Cl2 and cis-Pt(II)(benzothiazole)2Cl2 in cultured epimastigote forms of Trypanosoma cruzi. Studies have been performed at the ultrastructural level and the inhibitory effect of these complexes on macromolecule synthesis, evaluated by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation, has been investigated. DDP at concentrations of 50 and 100 micrograms/ml does not inhibit significantly the incorporation of radioactive precursors, but a clear decrease was observed with the 2 analogs. Eight hours of treatment at a concentration of 10 micrograms/ml rendered in all 3 cases an increase in autophagic vacuoles and lipids as well as an abnormal condensation of the nucleus chromatin.

The paper reports the synthesis, the chemical characterization and the IR data of new Pt(II) and Pt(IV) complexes, as well as their cytostatic activities on KB cells and antitumour properties against three tumour systems (P388 and L1210 leukemias and advanced B16 melanoma). The following ligands were used: 2,5-dichloroaniline, 3,4-dichloroaniline, 2,4,6-trichloroaniline, 3,4,5-trichloroaniline, 2,3,4,5-tetrachloroaniline and 2,3,5,6-tetrachloroaniline. The tri- and tetrachloroaniline-Pt(II) complexes displayed a fairly good antileukemic activity but lower than cisplatin. The effect of these compounds against advanced B16 melanoma appears more interesting. They show an activity comparable or in some cases higher than cisplatin and other 1,2-diaminocyclohexane-Pt(II) complexes. The M.O. Huckel's calculations were performed on the ligand molecules in order to help us to draw a structure-activity relationship for new compounds.