Subcurative Doses Research Articles

Phase 2 pilot trial to optimise pharmacometric evaluations in Chagas disease (CHARM: CHAgas disease PharMacometrics)

Chagas disease is a neglected disease caused by Trypanosoma cruzi that currently affects about 7 million people in the Americas and the American diaspora. Only two drugs are available to treat Chagas disease, both are associated with a high incidence of adverse events, particularly in adults, and they are also complex to administer. New treatments are needed urgently, but there is no established method of evaluating new drugs. We describe the study protocol for a pilot phase 2 descriptive observational study of parasite dynamics within the host, followed by a randomised evaluation of the pharmacokinetic-pharmacodynamic properties of subcurative doses of anti-chagasic drugs in patients with chronic Chagas disease. This study is divided into three stages. The general objective of the study is to characterise the dynamics of the parasite within the host at steady state and the pharmacokinetic-pharmacodynamic relationships for each of the three drugs studied. This is based on characterising the structure and parameters of a pharmacodynamic model of parasite turnover within the host. Each stage of the study targets specific key parameters in the pharmacodynamic model of parasite dynamics (both the blood stage and the tissue stage) for individual research participants. The primary outcome measure for all three stages is blood-stage parasite density, measured by quantitative PCR. Upon completion, research participants will receive definitive treatment for Chagas disease in accordance with national guidelines. Registration ISRCTN (ISRCTN26467068; 01/07/2021).

PD-L1 Immune Checkpoint Targeted Photoactivable Liposomes (iTPALs) Prime the Stroma of Pancreatic Tumors and Promote Self-Delivery.

Desmoplasia in pancreatic ductal adenocarcinoma (PDAC) limits the penetration and efficacy of therapies. It has been previously shown that photodynamic priming (PDP) using EGFR targeted photoactivable multi-inhibitor liposomes remediates desmoplasia in PDAC and doubles overall survival. Here, bifunctional PD-L1 immune checkpoint targeted photoactivable liposomes (iTPALs) that mediate both PDP and PD-L1 blockade are presented. iTPALs also improve phototoxicity in PDAC cells and induce immunogenic cell death. PDP using iTPALs reduces collagen density, thereby promoting self-delivery by 5.4-fold in collagen hydrogels, and by 2.4-fold in syngeneic CT1BA5 murine PDAC tumors. PDP also reduces tumor fibroblast content by 39.4%. Importantly, iTPALs also block the PD-1/PD-L1 immune checkpoint more efficiently than free α-PD-L1 antibodies. Only a single sub-curative priming dose using iTPALs provides 54.1% tumor growth inhibition and prolongs overall survival in mice by 42.9%. Overall survival directly correlates with the extent of tumor iTPAL self-delivery following PDP (Pearson's r = 0.670, p = 0.034), while no relationship is found for sham non-specific IgG constructs activated with light. When applied over multiple cycles, as is typical for immune checkpoint therapy, PDP using iTPALs promises to offer durable tumor growth delay and significant survival benefit in PDAC patients, especially when used to promote self-delivery of integrated chemo-immunotherapy regimens.

Lenalidomide Enhances Anti-BCMA Chimeric Antigen Receptor T Cell Function Against Multiple Myeloma

The development of chimeric antigen receptor (CAR) T cells that target antigens expressed on the surface of malignant plasma cells is a promising new treatment approach for relapsed refractory multiple myeloma patients. Although early clinical trials investigating single agent anti-B cell maturation antigen (BCMA) CAR-T cells achieved clinical responses in myeloma, additional combinatorial therapies may increase the effectiveness of this treatment. Lenalidomide, a small molecule immunomodulatory agent approved for use in multiple myeloma, can have both direct tumoricidal and T cell modulatory effects. Recent publications have highlighted the ability of lenalidomide to enhance T cell and CAR-T function in preclinical models.The immunomodulatory effects of lenalidomide were investigated in combination with an autologous cellular drug product transduced to express a BCMA-specific CAR with 4-1BB costimulatory and CD3 zeta endodomains. The intrinsic effects mediated by lenalidomide on CAR-T cell function were evaluated using antigen-coated beads labeled with varying concentrations of BCMA, allowing investigation into how stimulus strength and lenalidomide concentration influence CAR-T cell functionality. Lenalidomide increased CAR-T cell cytokine production across stimulation conditions and CAR-T cells exposed to low levels of antigen demonstrated the most marked increase in cytokine production and activation with the addition of lenalidomide in culture. To further investigate the effect of lenalidomide upon CAR-T cell stimulation, RNA-seq and ATAC-seq molecular analysis of CAR-T cells was carried out after 24 hours or 7 days of stimulation on BCMA coated beads with or without lenalidomide. Differential expression and accessibility results demonstrated an enriched involvement of immune synapse-associated genes, cytokine signaling, and T cell activation pathways consistent with the functional gains measured in the presence of lenalidomide.The effect of lenalidomide on CAR-T cell cytolytic activity, cytokine production, and proliferation was further evaluated in co-culture with multiple myeloma cell lines displaying a range of BCMA expression. The addition of lenalidomide during co-culture increased CAR-T cell effector cytokine production and cytolytic activity. Lenalidomide also increased CAR-T cell performance over 4 weeks in a repeated serial stimulation of BCMA CAR-T cells with MM1.S cells, an assay designed to predict CAR-T drug product fitness. The lenalidomide-associated increase in persistence and proliferation correlated with an increase in IL-2 production and activation marker expression at multiple time points following repeated stimulation. Next, to measure effector function after chronic stimulation, CAR-T cells were cultured for 7 days with BCMA beads, debeaded, and then cultured with multiple myeloma cell lines. Pre-stimulation on BCMA beads decreased CAR-T cell cytolytic activity and cytokine production in this assay. The addition of lenalidomide during CAR-T cell pre-stimulation increased activation marker expression, decreased PD-1 expression, and prevented the decrease in cytolytic activity relative to vehicle pre-stimulated CAR-T cells.To evaluate the in vivo activity of daily administration of lenalidomide in combination with BCMA CAR-T cells, NSG mice were injected intravenously with OPM2 multiple myeloma cells. Two dosing strategies were evaluated to determine the effect of dosing lenalidomide concurrently with CAR-T administration or two weeks following CAR-T administration. Both lenalidomide dosing schedules enhanced the function of sub-curative doses of CAR-T cells, as demonstrated by decreased tumor burden and increased animal survival.Overall, the addition of lenalidomide to BCMA CAR-T cells increased CAR-T function including activation, proliferation, cytolytic activity, and cytokine production in a concentration-dependent manner. The lenalidomide-induced increase in CAR-T function may be advantageous in the myeloma tumor microenvironment as a variety of factors can limit CAR-T efficacy including CAR-T exhaustion and low antigen density expressed on the tumor. Taken together, these data suggest that lenalidomide may potentially increase CAR-T cell activity upon encounter with surface expressed BCMA to enhance an anti-tumor response. DisclosuresWorks:Juno Therapeutics: Employment, Equity Ownership. Soni:Juno Therapeutics: Employment, Equity Ownership. Hauskins:Juno Therapeutics: Employment, Equity Ownership. Sierra:Juno Therapeutics: Employment, Equity Ownership. Baturevych:Juno Therapeutics: Employment, Equity Ownership. Jones:Juno Therapeutics: Employment, Equity Ownership. Curtis:Juno Therapeutics: Employment, Equity Ownership. Johnstone:Juno Therapeutics: Employment, Equity Ownership. Kugler:Juno Therapeutics: Employment, Equity Ownership. Jiang:Juno Therapeutics: Employment, Equity Ownership. Hause:Juno Therapeutics: Employment, Equity Ownership. Sather:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment, Equity Ownership. Ports:Juno Therapeutics: Employment, Equity Ownership.

Studies on resistance of Schistosoma to praziquantel XVIII Sensitivity to praziquantel in filial generations of praziquantel-resistant and -sensitive Schistosoma japonicum mixed infections

To investigate the sensitivity of adult worms of filial generations from praziquantel-resistant and -sensitive Schistosoma japonicum mixed infections to praziquantel. Mice were infected with the cercariae of an experimentally generated praziquantel-resistant S. japonicum isolate [median effective dose (ED50) = 277.4 mg/kg] and a laboratory-maintained praziquantel-sensitive S. japonicum isolate (ED50 = 99.6 mg/kg) at a mixture ratio of 1:1 and 2:1, which was maintained in the laboratory via the mouse-snail cycle for 8 generations. Then, mice were infected with the cercariae of the 8th filial-generation parasite, and grouped 35 days post-infection. Mice in the 5 treatment groups were given praziquantel treatment by gavage at a single oral dose of 37.5, 75, 150, 300 mg/kg and 600 mg/kg, while animals in the control group was administered orally with 2.5% cremophor EL. All mice were sacrificed 14 days post-treatment and adult worms were collected by perfusion of the portal vein. The worm burden reductions and praziquantel ED50 values were calculated. The praziquantel-resistant S. japonicum isolate generated from experimental induction with 12 rounds of praziquantel treatment with sub-curative doses was maintained in the laboratory via the mouse-snail cycle, and mice were infected with the cercariae of the 8th filial-generation parasite. The praziquantel ED50 value against the 8th filial-generation adults was measured. After mice were infected with the mixture of cercariae of PZQ-resistant and -sensitive S. japonicum isolates at a ratio of 1:1, the praziquantel ED50 was 135.2 mg/kg against the adults of the 8th filial-generation parasite. After mice were infected with the mixture of cercariae of PZQ-resistant and -sensitive S. japonicum isolates at a ratio of 2:1, the praziquantel ED50 was 129.2 mg/kg against the adults of the 8th filial-generation parasite. In addition, the praziquantel ED50 was 208.4 mg/kg against the adults of the 8th filial-generation S. japonicum without the selection pressure of praziquantel. Compared with the experimentally induced praziquantel-resistant S. japonicum isolate, the adult worms of the filial-generation S. japonicum show a reduced sensitivity to praziquantel in the same host following infection with the mixture of cercariae of praziquantel-resistant and -sensitive S. japonicum isolates. The adult worms of the filial generation of the praziquantel-resistant S. japonicum isolate without the selection pressure of praziquantel may still maintain the resistance to praziquantel.

Artemisinin exposure at the ring or trophozoite stage impacts Plasmodium falciparum sexual conversion differently

Malaria transmission is dependent on the formation of gametocytes in the human blood. The sexual conversion rate, the proportion of asexual parasites that convert into gametocytes at each multiplication cycle, is variable and reflects the relative parasite investment between transmission and maintaining the infection. The impact of environmental factors such as drugs on sexual conversion rates is not well understood. We developed a robust assay using gametocyte-reporter parasite lines to accurately measure the impact of drugs on sexual conversion rates, independently from their gametocytocidal activity. We found that exposure to subcurative doses of the frontline antimalarial drug dihydroartemisinin (DHA) at the trophozoite stage resulted in a ~ fourfold increase in sexual conversion. In contrast, no increase was observed when ring stages were exposed or in cultures in which sexual conversion was stimulated by choline depletion. Our results reveal a complex relationship between antimalarial drugs and sexual conversion, with potential public health implications.

Abstract 2889: SGN-CD30C, a new CD30-directed camptothecin antibody-drug conjugate (ADC), shows strong anti-tumor activity and superior tolerability in preclinical studies

Abstract SGN-CD30C, a new CD30-directed ADC, utilizes a potent camptothecin derivative as the cytotoxic payload. SGN-CD30C targets the same antigen as brentuximab vedotin (BV), though the payload has a different mechanism of action, namely inhibiting topoisomerase I rather than disrupting microtubules. Unlike monomethyl auristatin E, camptothecin-based therapies do not cause peripheral neuropathy clinically, suggesting that SGN-CD30C may have the potential to avoid one of the most common adverse events associated with BV. In preclinical studies, SGN-CD30C demonstrated strong monotherapy activity, inducing durable tumor regressions in multiple lymphoma models and eliciting cures in a BV-resistant tumor model. Furthermore, bystander killing of antigen-negative tumor cells, which is thought to be an important attribute of BV, was confirmed in vivo using CD30-heterogeneous tumors. SGN-CD30C was well-tolerated in non-human primate (NHP) toxicology studies and demonstrated ~6-fold higher maximum tolerated dose (MTD) when compared to BV. The primary toxicity for SGN-CD30C in NHP studies was anemia due primarily to bone marrow suppression of erythropoiesis. SGN-CD30C had no effect on neutrophil counts and caused only minimal to mild decreases in platelets. The lack of significant neutropenia seen with SGN-CD30C contrasts with BV, indicating the two antibody-drug conjugates may have non-overlapping dose limiting toxicities. We theorized using a reduced dose of BV in combination with SGN-CD30C could provide a dual mechanism of action to retain or enhance anti-tumor activity while decreasing the incidence of non-overlapping toxicities (i.e. peripheral neuropathy). To test this hypothesis preclinically, we evaluated the anti-tumor activity of BV and SGN-CD30C at sub-curative doses in a Karpas-299 xenograft model. The results show the combination of SGN-CD30C + BV generated a higher number of durable CRs compared to the equivalent monotherapy doses, thereby confirming strong anti-tumor activity by using a lower dose of each ADC in combination. In summary, our data show SGN-CD30C is a compelling therapeutic candidate for CD30-positive malignancies. The strong anti-tumor activity and superior tolerability suggest SGN-CD30C has a favorable therapeutic window. Furthermore, the distinct mechanism of action and non-overlapping toxicity profile offers the potential to combine SGN-CD30C with BV for enhanced risk/benefit. Citation Format: Maureen Ryan, Ryan Lyski, Lauren Bou, David Meyer, Steven Jin, Jessica Simmons, Dennis Benjamin, Peter Senter, Scott Jeffrey. SGN-CD30C, a new CD30-directed camptothecin antibody-drug conjugate (ADC), shows strong anti-tumor activity and superior tolerability in preclinical studies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2889.

Feasibility of Combination Intra-arterial Yttrium-90 and Irinotecan Microspheres in the VX2 Rabbit Model.

To evaluate the combination of 90Y radioembolization (Y90) and drug-eluting bead irinotecan (DEBIRI) microspheres in the VX2 rabbit model. An initial dose finding study was performed in 6 White New Zealand rabbits to identify a therapeutic but subcurative dose of Y90. In total, 29 rabbits were used in four groups: Y90 treatment (n = 8), DEBIRI treatment (n = 6), Y90 + DEBIRI treatment (n = 7), and an untreated control group (n = 8). Hepatic toxicity was evaluated at baseline, 24h, 72h, 1week, and 2weeks. MRI tumor volume (TV) and enhancing tumor volume were assessed baseline and 2weeks. Tumor area and necrosis were evaluated on H&E for pathology. Infused activities of 84.0-94.4MBq (corresponding to 55.1-72.7Gy) were selected based on the initial dose finding study. Infusion of DEBIRI after Y90 was technically feasible in all cases (7/7). Overall, 21/29 animals survived to 2weeks, and the remaining animals had extrahepatic disease on necropsy. Liver transaminases were elevated with Y90, DEBIRI, and Y90 + DEBIRI compared to control at 24h, 72h, and 1week post-treatment and returned to baseline by 2weeks. By TV, Y90 + DEBIRI was the only treatment to show statistically significant reduction at 2weeks compared to the control group (p = 0.012). The change in tumor volume (week 2-baseline) for both Y90 + DEBIRI versus control (p = 0.002) and Y90 versus control (p = 0.014) was significantly decreased. There were no statistically significant differences among groups on pathology. Intra-arterial Y90 + DEBIRI was safe and demonstrated enhanced antitumor activity in rabbit VX2 tumors. This combined approach warrants further investigation.

Impact of Single Dose Photons and Carbon Ions on Perfusion and Vascular Permeability: A Dynamic Contrast-Enhanced MRI Pilot Study in the Anaplastic Rat Prostate Tumor R3327-AT1.

We collected initial quantitative information on the effects of high-dose carbon (12C) ions compared to photons on vascular damage in anaplastic rat prostate tumors, with the goal of elucidating differences in response to high-LET radiation, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Syngeneic R3327-AT1 rat prostate tumors received a single dose of either 16 or 37 Gy 12C ions or 37 or 85 Gy 6 MV photons (iso-absorbed and iso-effective doses, respectively). The animals underwent DCE-MRI prior to, and on days 3, 7, 14 and 21 postirradiation. The extended Tofts model was used for pharmacokinetic analysis. At day 21, tumors were dissected and histologically examined. The results of this work showed the following: 1. 12C ions led to stronger vascular changes compared to photons, independent of dose; 2. Tumor growth was comparable for all radiation doses and modalities until day 21; 3. Nonirradiated, rapidly growing control tumors showed a decrease in all pharmacokinetic parameters (area under the curve, Ktrans, ve, vp) over time; 4. 12C-ion-irradiated tumors showed an earlier increase in area under the curve and Ktrans than photon-irradiated tumors; 5. 12C-ion irradiation resulted in more homogeneous parameter maps and histology compared to photons; and 6. 12C-ion irradiation led to an increased microvascular density and decreased proliferation activity in a largely dose-independent manner compared to photons. Postirradiation changes related to 12C ions and photons were detected using DCE-MRI, and correlated with histological parameters in an anaplastic experimental prostate tumor. In summary, this pilot study demonstrated that exposure to 12C ions increased the perfusion and/or permeability faster and led to larger changes in DCE-MRI parameters resulting in increased vessel density and presumably less hypoxia at the end of the observation period when compared to photons. Within this study no differences were found between curative and sub-curative doses in either modality.

Abstract 2320: Avadomide (CC-122) increases effector function and reverses exhaustion in chronically stimulated lisocabtagene maraleucel anti-CD19 CAR T drug product

Abstract Lisocabtagene maraleucel (liso-cel) is an investigational drug product composed of autologous CD8+ and CD4+ T cells expressing a CD19-specific chimeric antigen receptor (CAR), being evaluated in clinical trials for treatment of B cell malignancies. The CELMoD CC-122 is being evaluated in non-Hodgkin lymphoma (NHL) and can augment T cell function through Cereblon-mediated degradation of Ikaros and Aiolos. Combination approaches aimed at enhancing CAR T cell function may increase the rate, depth, and durability of clinical responses. Here we report pre-clinical results supporting the potential combination of CC-122 an liso-cel. To determine potentiating effects of CC-122 following acute activation (anti-idiotypic Ab or CD19+ targets), T cells expressing the CAR of liso-cel were cultured with CC-122 and assessed for intracellular signaling, cytolytic activity, cytokine production, and activation marker expression. CC-122 significantly increased NFκB, Nur77, and STAT5 as well as effector cytokine production and activation marker expression. Activation-induced exhaustion of CAR T may decrease durable responses in patients. We developed a chronic stimulation assay to render CAR T cells hypofunctional (reduced cytolysis and IL-2 secretion) and examined whether enhanced activation mediated by CC-122 would exhaust the CD19-directed CAR T cells more rapidly. CC-122 was added in one of two regimens: during chronic stimulation (“concurrent”) or during re-challenge (“rescue”). Effects on exhaustion in “concurrent” or “rescue” cultures were evaluated, including by RNAseq and measuring of effector function in re-challenge assays with CD19+ targets. Surprisingly, “concurrent” CC-122 partially reversed a gene signature associated with CAR T cell hypofunctionality and preserved more effector function relative to controls. In “rescue” assays, CC-122 dose-dependently increased cytokine production and restored cytolytic function of exhausted CAR T cells upon re-challenge with CD19+ tumor spheroids. In a disseminated tumor xenograft model, concurrent or delayed dosing of CC-122, in combination with two sub-curative doses of liso-cel, significantly increased median survival by 30 to 40 days. In some cases, survival endpoints were not reached with the combination. Taken together, CC-122 treatment of CD19-directed CAR T cells was shown to enhance CAR T activation in acute stimulation assays; limit CAR T exhaustion onset during chronic stimulation as assessed by gene expression; and reverse an exhausted, hypofunctional CAR T state induced by chronic stimulation. The data suggest that liso-cel pharmacologic activity may both be enhanced and extended by this combinatorial approach. These findings support an ongoing clinical investigation evaluating the addition of CC-122 to liso-cel in patients with R/R, aggressive NHL (NCT03310619). Citation Format: Heidi K. Jessup, Evan P. Thomas, Jim S. Qin, Yue Jiang, Oleksandr Baturevych, Neha Soni, Aye T. Chen, Ruth A. Salmon, Melissa G. Works, Michael O. Ports. Avadomide (CC-122) increases effector function and reverses exhaustion in chronically stimulated lisocabtagene maraleucel anti-CD19 CAR T drug product [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2320.

Combinatorial Effects of Monensin in Liposome Formulations with Antimalarial Drugs Against Blood Stages of Plasmodium falciparum in Culture and P. berghei Infection

Background: Monensin is a broad spectrum polyether antibiotic produced by Streptomyces cinnamonensis. It is widely used as veterinary medicine in the treatment of coccidiosis. Objective: The goal of this study was to assess the antimalarial efficacy of monensin in liposomal form in combination with potent antimalarial drugs like chloroquine, piperaquine, or FR900098 in free form under in vitro and in vivo condition. Method: In vitro drug sensitivity assay for different drug combinations against blood stages of chloroquine susceptible (3D7) and chloroquine resistant (INDO) strains of Plasmodium falciparum in culture was assessed by [3H]-hypoxanthine incorporation assay. In vivo efficacy was evaluated using the standard 4-day Peters’ test in established mice models of both Plasmodium berghei NK65 and lethal strain of P. berghei ANKA. Results: Our results indicate that monensin exhibits superior efficacy than partner antimalarial drugs tested. The drug combinations between monensin/chloroquine, monensin/ piperaquine, and monensin/FR900098 showed synergistic to additive action, at their 50- percent inhibitory concentrations (IC50s) against P. falciparum strains in culture. Monensin in liposomal formulations in combination with subcurative doses of partner antimalarial drugs effectively reduced parasitic burden in P. berghei infected mice than comparable doses of antimalarial drugs used alone. Conclusion: Our results demonstrate that liposomal monensin in combination with partner antimalarials (chloroquine, piperaquine or FR900098) showed positive interactions with enhanced parasite killing on P. falciparum strains and P. berghei infection in murine model. This approach may provide a promising chemotherapeutic regimen at lower doses for treating human malarial infections. Keywords: Drug combinations, liposome monensin, plasmodium, antimalarials, chemotherapy.

Targeted Delivery of Auristatin-Modified Toxins to Pancreatic Cancer Using Aptamers.

Pancreatic cancer is one of the most lethal malignancies. Treatment with the first-line agent, gemcitabine, is often unsuccessful because it, like other traditional chemotherapeutic agents, is non-specific, resulting in off-target effects that necessitate administration of subcurative doses. Alternatively, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are highly toxic small molecules that require ligand-targeted delivery. MMAE has already received FDA approval as a component of an anti-CD30 antibody-drug conjugate, brentuximab vedotin. However, in contrast to antibodies, aptamers have distinct advantages. They are chemicals, which allows them to be produced synthetically and facilitates the rapid development of diagnostics and therapeutics with clinical applicability. In addition, their small size allows for enhanced tissue distribution and rapid systemic clearance. Here, we assayed the toxicity of MMAE and MMAF conjugated to an anti-transferrin receptor aptamer, Waz, and an anti-epidermal growth factor receptor aptamer, E07, on the pancreatic cancer cell lines Panc-1, MIA PaCa-2, and BxPC3. In vitro, our results indicate that these aptamers are a viable option for the targeted delivery of toxic payloads to pancreatic cancer cells.

Antileishmanial Activity of Pyrazolopyridine Derivatives and Their Potential as an Adjunct Therapy with Miltefosine.

A series of pyrazolo(dihydro)pyridines was synthesized and evaluated for antileishmanial efficacy against experimental visceral leishmaniasis (VL). Among all compounds, 6d and 6j exhibited better activity than miltefosine against intracellular amastigotes. Compound 6j (50 mg/kg/day) was further studied against Leishmania donovani/BALB/c mice via the intraperitoneal route for 5 days and displayed >91 and >93% clearance of splenic and liver parasitic burden, respectively. Combination treatment of 6j with a subcurative dose of miltefosine (5 mg/kg) in BALB/c mice almost completely ameliorated the disease (>97% inhibition) by augmenting nitric oxide generation and shifting the immune response toward Th1. Furthermore, investigating the effect of 6j on Leishmania promastigotes revealed that it induced molecular events, such as a loss in mitochondrial membrane potential, externalization of phosphatidylserine, and DNA fragmentation, that ultimately resulted in the programmed cell death of the parasite. These results along with pharmacokinetic studies suggest that 6j could be a promising lead for treating VL as an adjunct therapy with miltefosine.

15d-Prostaglandin J2 induced reactive oxygen species-mediated apoptosis during experimental visceral leishmaniasis.

15-Deoxy-delta (12,14)-prostaglandin J2 (15d-PgJ2) is a potent bioactive lipid mediator, known to possess several roles in cell regulation and differentiation along with antimicrobial efficacy against different bacterial and viral infections. In the present study, we investigated the therapeutic efficacy and mechanism of action of 15d-PgJ2 in vitro in Leishmania donovani promastigotes and infected J774 macrophages, and in vivo in Balb/c mice/golden hamster model of experimental visceral leishmaniasis. 15d-PgJ2 effectively killed L. donovani promastigotes and amastigotes in vitro with IC50 of 104.6 and 80.09nM, respectively. At 2mg/kg (mice) and 4mg/kg (hamster) doses, 15d-PgJ2 decreased >90% spleen and liver parasite burden. It significantly reduced interleukin (IL)-10 and transforming growth factor (TGF)-β synthesis in infected macrophages and splenocytes. 15d-PgJ2 induced reactive oxygen species (ROS)-dependent apoptosis of promastigotes by triggering phosphatidyl serine externalization, mitochondrial membrane damage and inducing caspase-like activity. In vitro drug interaction studies revealed an indifference to the synergistic association of 15d-PgJ2 with Miltefosine and Amphotericin-B (Amp-B). Moreover, when combined with sub-curative doses of Miltefosine and Amphotericin-B, 15d-PgJ2 resulted in >95% parasite removal. Our results suggested that 15d-PgJ2 induces mitochondria-dependent apoptosis of L. donovani and is a good therapeutic candidate for adjunct therapy against experimental visceral leishmaniasis. 15d-PgJ2 effectively eliminated both promastigotes and amastigotes form of L. donovani. 15d-PgJ2 decreased parasite burden from infected mice and hamsters with reduced Th2 cytokines. 15d-PgJ2 induced ROS-mediated mitochondrial apoptosis of L. donovani promastigotes. 15d-PgJ2 is a good therapeutic candidate for adjunct therapy with Miltefosine and Amp-B.

Transmission electron microscopic observations on ultrastructural alterations in Schistosoma mansoni adult worms recovered from C57BL/6 mice treated with radiation-attenuated vaccine and/or praziquantel in addition to passive immunization with normal and vaccinated rabbit sera against infection.

Although the current treatment of schistosomiasis relies largely on praziquantel (PZQ), it has not been successful in significantly reducing the overall rate of disease cases, one of the suggested reasons being the inevitable resistance to PZQ. Previous studies showed that radiation-attenuated vaccine provides protection against Schistosoma mansoni in a host of various species. In the present study, we evaluated the effect of various vaccination strategies in C57BL/6 mice, including single or multiple vaccination strategy, subcurative dose (20 mg/kg) of PZQ, and a combination of single vaccination with subcurative dose of PZQ. Treatment either with subcurative dose of PZQ or with a single vaccination of attenuated cercariae (500 per mouse), caused significant reduction in total worm burden, hepatic, and intestinal ova counts of 43.03, 73.2, and 59.5 and 37.97, 52.02, and 26.3%, respectively. Furthermore, tegumental changes were observed. In multiple vaccinated group, there was an extensive lysis in tegumental layers. High deformations in gastrodermis, testis cells, vitelline cells, and oocytes were recorded. Also, this study is to explore the role of humoral immunity using highly resistant rabbits that had been exposed to three immunizations with ultraviolet (UV)-irradiated cercariae (8000 per rabbit in each immunization), and their sera were tested for their ability to transfer protection. The reduction in challenge worm burden had reached 32.76-43.64% when compared with recipients of normal serum or no serum. The reduction in hepatic and intestinal ova counts reached to 74.4 and 71.08% in group immunized with vaccinated rabbit sera. Swelling and extensive lysis of tegumental layers, gastrodermis lumen, spermatocytes, and deformation of oocytes were recorded with more severity than that recorded in normal rabbit sera group. Our findings recorded that multiple vaccination strategy is the most effective strategy then passive transfer of vaccinated rabbit. This gives guiding in the design the appropriate therapeutic strategy.

In vitro and in vivo anti-malarial activity of tigecycline, a glycylcycline antibiotic, in combination with chloroquine.

BackgroundSeveral antibiotics have shown promising anti-malarial effects and have been useful for malarial chemotherapy, particularly in combination with standard anti-malarial drugs. Tigecycline, a semi-synthetic derivative of minocycline with a unique and novel mechanism of action, is the first clinically available drug in a new class of glycylcycline antibiotics.MethodsTigecycline was tested in vitro against chloroquine (CQ)-sensitive (D6) and resistant strains (W2) of Plasmodium falciparum alone and in combination with CQ. Tigecycline was also tested in vivo in combination with CQ in Plasmodium berghei-mouse malaria model for parasitaemia suppression, survival and cure of the malaria infection.ResultsTigecycline was significantly more active against CQ-resistant (W2) than CQ-susceptible (D6) strain of P. falciparum. Tigecycline potentiated the anti-malarial action of CQ against the CQ-resistant strain of P. falciparum by more than seven-fold. Further, treatment of mice infected with P. berghei with tigecycline (ip) produced significant suppression in parasitaemia development and also prolonged the mean survival time. Treatment with as low as 3.7 mg/kg dose of tigecycline, once daily for four days, produced 77-91% suppression in parasitaemia. In vivo treatment with tigecycline in combination with subcurative doses of CQ produced complete cure in P. berghei-infected mice.ConclusionResults indicate prominent anti-malarial action of tigecycline in vitro and in vivo in combination with CQ and support further evaluation of tigecycline as a potential combination candidate for treatment of drug-resistant cases of malaria.

Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention

To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.

Abstract LB-244: BAY 87-2243, an inhibitor of HIF-1α - induced gene activation, showed promising anti-tumor efficacy in combination with anti-angiogenic therapy and irradiation in preclinical tumor models

Abstract Hypoxia is a hallmark of solid tumors and is associated with local invasion, metastatic spread and resistance to radio- as well as chemotherapy (Vaupel et al., 2004). Furthermore, hypoxia constitutes an independent negative prognostic factor in a diverse range of malignant tumors (Harris, 2002; Vaupel et al., 2004). The cellular adaptation to hypoxia is mediated by a heterodimeric transcription factor hypoxia inducible factor-1 (HIF1). HIF-1α, one subunit of HIF, is constitutively degraded under normoxic conditions, but is stabilized and activated in hypoxic regions of tumors. Nuclear activated HIF-1 controls the expression of >100 genes involved in cellular energy metabolism, neoangiogenesis, anti-apoptotic and pro-proliferative mechanisms promoting invasion and metastasis. Recently, we showed that BAY 87-2243, a small molecule inhibitor of hypoxia-induced HIF-1 activation that specifically suppressed hypoxia-induced HIF-1α stabilization is able to achieve very promising results in combination with standard treatments in several preclinical tumor xenograft models. Here, we present outstanding preclinical anti-tumor efficacy of BAY 87-2243 in combination with anti-angiogenic therapy and irradiation in xenografts of various histological type. BAY 87-2243 was dosed continuously in combination with the tyrosine kinase inhibitors regorafenib as well as sorafenib and the anti-VEGF antibody, bevacizumab. The combinations produced long-term tumor growth control or tumor stasis of either cell line derived or patient-derived subcutaneous colorectal carcinoma xenografts (HCT116, HT29, LoVo). In a fully established lung carcinoma model (A549) the combination treatment of BAY 87-2243 with regorafenib resulted in slight regression of tumor growth that was maintained for up to 18 days. In a prostate carcinoma model (PC-3) a combination of BAY 87-2243 and regorafenib resulted in an additive improvement of the tumor growth inhibition as compared to either agents alone. A first combination treatment of BAY 87-2243 and a single subcurative irradiation dose (8 Gy) in a radioresistant, severely hypoxic head and neck squamous cell carcinoma xenograft (UT-SCC-5) led to tumor regression, which did not occur in the respective single-agent/radiation treatment. These promising preclinical results suggest that inhibition of HIF-1 by BAY 87-2243 in particular in combination with anti-angiogenic therapy and irradiation is an innovative approach to cancer therapy with the potential to overcome hypoxia/HIF-induced tumor resistance mechanisms. Harris AL (2002) Hypoxia-a key regulatory factor in tumor growth. Nat Rev Cancer 2: 38-47 Vaupel P, Mayer A, Hockel M (2004) Tumor hypoxia and malignant progression. Methods Enzymol 381:335-354 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-244. doi:1538-7445.AM2012-LB-244

Augmentation of antileishmanial efficacy of miltefosine in combination with tuftsin against experimental visceral leishmaniasis

Current drugs for the treatment of visceral leishmaniasis are inadequate, and their efficacies are also compromised due to suppression of immune function during the course of infection. Miltefosine is the only promising orally active antileishmanial drug, but due to its long half-life, there is risk of development of resistance. To overcome these problems, efforts are needed to develop combination therapy of miltefosine with effective immunostimulating agents where a decrease of parasitic burden and simultaneous enhancement of adaptive immunity can be achieved. In the present study, we have explored the antileishmanial efficacy of a subcurative dose of miltefosine in combination with free as well as liposomal palmitoyl tuftsin (p-tuftsin) using a Leishmania donovani/BALB/c mouse model. When miltefosine (2.5 mg/kg for 5 days) was given with free p-tuftsin, the inhibitory effect was significantly increased from 49.6% to 66% (P < 0.01), which was further enhanced up to 81% (P < 0.001) when given after liposomal encapsulation of p-tuftsin. Significant enhancement in parasitic inhibition (93%, P < 0.01) was witnessed when animals were co-administered with liposomal p-tuftsin + 5 mg/kg × 5 days dose of miltefosine (72.1%). Enhancement in the production of Th1 cytokines (IL-12, TNF-α, and IFN-γ), reactive oxygen, and nitrogen metabolites was witnessed in the combination group. A remarkable increase in phagocytosis index was also observed indicating overall immunological enhancement to antileishmanial activity of miltefosine by p-tuftsin.

Effect of a combination of the new antischistosomal drug Ro 15-5458 and praziquantel on different strains of Schistosoma mansoni infected mice.

The possible additive or synergistic effects of both praziquantel (CAS 55268-74-1) and a new antischistosomal drug, Ro 15-5458 (10-(2-diethylamino)thyl)-9-acridanone(thiazolidin-2-yl-i dene)hydrazone, CAS 92928-47-7) were studied in two different strains of Schistosoma mansoni infected mice, namely CD susceptible and SO4 resistant strains. Assessment of cure was performed using the following parameters: hepatic and intestinal tissue egg load and distribution, oogram changes in the small intestine and histopathological examination of the mice livers. In this study, a combination was used between 1/3 the curative doses of praziquantel and Ro 15-5458. This combination therapy proved to be beneficial as regards the percentage parasite reduction and hepatic worm shift (99.4% and 100%, respectively, in the CD susceptible mouse strains, compared to 84.1% and 34.8% in the SO4 resistant strains). Treatment with subcurative doses of praziquantel and Ro 15-5458 resulted in 78.6% intestinal dead ova and 21.4% mature ones. This score shifted to 98.6% and 1.4% dead and mature ova, respectively, in the SO4 resistant strains. Again the range of liver granulomata in the CD susceptible and SO4 resistant strains receiving subcurative doses of both drugs was 4-6 and 2-5, respectively, in five successive low power fields, while in the infected untreated control mice, this range reached 8-11 and 5-9, respectively. Histopathological sections of the liver revealed a small fibrocellular granuloma with few inflammatory cells and excess fibrous collagen tissue deposition in animals undergoing the combination therapy. This contrasts with the large fibrocellular granulomata seen in the infected untreated control mice. These results may be of value in endemic areas of schistosomiasis, due to the unexpected emergence of drug resistance against the currently used antischistosomal drug, praziquantel in these areas.

Effect of Pam3Cys induced protection on the therapeutic efficacy of miltefosine against experimental visceral leishmaniasis

Prophylactic potential of synthetic bacterial lipopeptide and a TLR2 agonist, Pam3Cys was first evaluated against experimental visceral leishmaniasis in rodent model. After establishing the potential its effect on therapeutic efficacy of miltefosine was also studied. Pam3Cys showed 74.64% inhibition in parasitic establishment when administered by ip route at a dose of 100μg/animal spaced at two weeks, i.e. on day −7 and +7 of challenge with Leishmania donovani amastigotes. However, when aforesaid dose of Pam3Cys was given with sub-curative dose of miltefosine (2.5mg/kg for 5 days) its efficacy enhanced from 49.80% to 92.25%. These findings revealed that this lipopeptide has potential protective efficacy which significantly enhanced the therapeutic efficacy of miltefosine used at low dose against Leishmania infection and warrants detailed investigations on its possible immunopotentiatory actions.