Nanoparticle-Hydrogel Systems Containing Platensimycin for Local Treatment of Methicillin-Resistant Staphylococcus aureus Infection.

Skin and soft tissue infections require effective and sustained topical administration. Platensimycin (PTM) is a natural drug lead that targets bacterial fatty acid synthases and has a great potential to treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA). To facilitate the use of PTM against local MRSA infections, we prepared polyacrylamide hydrogels containing polyamidoamine (PAMAM)/PTM nanoparticles (NP-gel(PTM)) for the controlled release of PTM. NP-gel(PTM) can continuously inhibit the growth of MRSA and its biofilm formation in simulated drug flow models in vitro. In situ implantation of NP-gel(PTM) could treat MRSA-infected subcutaneous soft tissues without toxicity. For MRSA-infected skin wounds, NP-gel(PTM) not only showed strong anti-MRSA activity but also accelerated more wound healing than the widely used antibiotic mupirocin. Collectively, PTM is expected to be used in this safe and effective NP-gel delivery platform for the treatment of local infections, which might help to alleviate the current antibiotic resistance crisis.

Differential effects of aquatic anaesthetics on the pharmacokinetics of antibiotics: Examples using florfenicol in Nile tilapia (Oreochromis niloticus).

Anaesthetics are commonly applied in pharmacokinetic (PK) studies to assure smooth handling of experimental procedures or to promote animal welfare. However, the influence of anaesthetics on the PK of co-administered drug is generally unknown but assumes ignorable. The goal of the study was to investigate the effect of tricaine methanesulfonate (MS-222), 2-phenoxyethanol (2-PE) and eugenol (EUG) on the PK of florfenicol (FF) in Nile tilapia. Twenty-eight fish were repeatedly exposed to 90 ppm EUG, 300 ppm MS-222 or 900 ppm 2-PE before FF oral administration (15 mg/kg) and each successive blood sampling. The serum concentration-time profiles were analysed by a 2-compartmental model, and the generated parameters in the control (without anaesthetic) and anaesthetic groups were statistically compared. The results demonstrated that the serum concentrations of each anaesthetic were similar at every FF sampling times (70 µg/ml for MS-222; 277 µg/ml for 2-PE; and 61 µg/ml for EUG). In comparison with the control group, the repeated use of MS-222 did not result in a statistical difference in most of the PK parameters. In contrast, the elimination half-lives of the 2-PE and EUG groups were significantly longer whereas the absorption and distribution half-lives of the 2-PE group were significantly shorter than the control, resulting in altered optimal dosages in the simulation modelling. Whether or not the numbers and extent of PK parameters change mitigate subsequent estimations of other PK-derived secondary values such as dosing regimen and withdrawal time remains to be elucidated, but the auxiliary use of anaesthetics in PK studies should not assume uninfluential.

A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile.

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (Ki = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC50 = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.

A suitable anaesthetic protocol for metamorphic zebrafish.

Zebrafish are frequently used as a means to investigate development. These studies increasingly require repeated anaesthesia of zebrafish during juvenile (i.e. metamorphic) stages. The effects of anaesthesia during this time remain poorly studied. The aim of this study was to develop a reliable method that can be used for frequently repeated anaesthesia during juvenile stages. Initially, we assessed different concentrations of MS-222, the most commonly used fish anaesthetic, for 30 minute anaesthesia with recovery. We showed that suitable MS-222 doses could be identified for the smallest (7mm) and largest (20mm) fish. However, we found that juvenile fish within a specific metamorphic window (sized between 8-16 mm) were vulnerable to MS-222 and no standard concentration of MS-222 provided reliable anaesthesia under these conditions. Hence we focussed our efforts on identifying a protocol for these stages. We tested six different published anaesthesia protocols P1-P6 where P1, P2 corresponds to 0.01% MS-222, P3, P4: 0.085% 2-phenoxyethanol and P5, P6: 0.00025%/0.0050% Propofol/Lidocaine. In protocols P1, P3, P5 fish were maintained by immersion, whilst in P2, P4 and P6: fish were maintained on an anaesthetic-doused cotton-pad. We assessed reliable anaesthesia using 10 fish for 10 minutes, with full recovery. Our data allowed us to eliminate two of these protocols as unsuitable for short term anaesthesia with recovery of juvenile fish. Extending these studies to explore repeated anaesthesia at 4 day intervals for 20 days under the remaining four protocols, we showed that P1 and P4 were both suitable for repeated anaesthesia, and that P4 was most suitable for imaging. We confirmed that P4 remained suitable when the frequency of anaesthesia was increased to every 2 days. We conclude that this protocol provides a refinement to the current protocol for repeated anaesthesia with recovery of juvenile zebrafish in the vulnerable metamorphic window.

A "ligand-targeting" peptide-drug conjugate: Targeted intracellular drug delivery by VEGF-binding helix-loop-helix peptides via receptor-mediated endocytosis.

As a new alternative to antibody-drug conjugates, we generated "ligand-targeting" peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.

Effects of anesthetic MS-222 on stress and reproduction of South American silver catfish (Rhamdia quelen) males.

Anesthesia is a common practice used in fish research and aquaculture. It is important to understand anesthetic effects on the animal and tissues of interest to ensure validity of data and to improve animal welfare in research and fish production endeavors. The production of some captive fish species is only possible by imposing artificial reproduction procedures, and manipulation of fish for these purposes is a stressor. The purpose of this study, therefore, was to evaluate effects of different concentrations (100, 200, and 300 mg/L) of the anesthetic MS-222 (tricaine methanesulfonate) on cortisol concentrations and effects on sperm quality in Rhamdia quelen. After hormonal induction of gamete production, 28 sexually mature males were randomly assigned to treatments, and milt and blood samples were collected. Anesthesia induction time, motility rate, sperm concentration and morphology, plasma cortisol concentrations, and reproductive hormone concentrations (testosterone, 17-α-hydroxyprogesterone, and estradiol) were evaluated. Sperm motility was greater in the control than 300 mg/L treatment group but did not differ among the control, 100, and 200 mg/L groups. The estradiol concentration was greater in non- anesthetized than anesthetized Rhamdia quelen, but plasma cortisol concentrations did not differ among treatment groups (182.50 ±â€¯42.03 ng/mL). The anesthetic MS-222 at concentrations of 100, 200, and 300 mg/L did not inhibit the stress response due to handling of Rhamdia quelen males. In addition, treatment with MS-222 was not effective in inhibiting detrimental effects on sperm quality because this treatment was associated with impaired sperm motility and lesser concentrations of plasma estradiol.

Controlled coupling of an ultrapotent auristatin warhead to cetuximab yields a next-generation antibody-drug conjugate for EGFR-targeted therapy of KRAS mutant pancreatic cancer.

BACKGROUND: Antibody-drug conjugate (ADC) construction poses numerous challenges that limit clinical progress. In particular, common bioconjugation methods afford minimal control over the site of drug coupling to antibodies. Here, such difficulties are overcome through re-bridging of the inter-chain disulfides of cetuximab (CTX) with auristatin-bearing pyridazinediones, to yield a highly refined anti-epidermal growth factor receptor (EGFR) ADC. METHODS: In vitro and in vivo assessment of ADC activity was performed in KRAS mutant pancreatic cancer (PaCa) models with known resistance to CTX therapy. Computational modelling was employed for quantitative prediction of tumour response to various ADC dosing regimens. RESULTS: Site-selective coupling of an auristatin to CTX yielded an ADC with an average drug:antibody ratio (DAR) of 3.9, which elicited concentration- and EGFR-dependent cytotoxicity at sub-nanomolar potency in vitro. In human xenografts, the ADC inhibited tumour growth and prolonged survival, with no overt signs of toxicity. Key insights into factors governing ADC efficacy were obtained through a robust mathematical framework, including target-mediated dispositional effects relating to antigen density on tumour cells. CONCLUSIONS: Together, our findings offer renewed hope for CTX in PaCa therapy, demonstrating that it may be reformatted as a next-generation ADC and combined with a predictive modelling tool to guide successful translation.

Arsenic trioxide and BIBR1532 synergistically inhibit breast cancer cell proliferation through attenuation of NF-κB signaling pathway.

AIMS: Despite the remarkable anti-proliferative effects of Arsenic trioxide (ATO) in breast cancer cells, the requirement of high, toxic concentrations to induce apoptosis may cause serious side effects in patients. In the present study, we aimed to use BIBR1532, an hTERT inhibitor, in combination with ATO to sensitize MCF7 and MDA-231 cells to lower concentrations of ATO. MAIN METHODS: Breast cancer cell lines MCF7 and MDA-231 were cultured and treated with different doses of ATO and BIBR1532 for 48 h and its effects on cell survival and proliferation were analyzed by MTT, crystal violet staining, colony formation assay, cell cycle, AnnexinV/PI and Real-time PCR tests. KEY FINDINGS: ATO and BIBR1532 synergistically inhibited proliferation and colony-forming ability of breast cancer cells. Besides, BIBR1532 augmented ATO-induced cytotoxic effects via triggering G1 cell cycle arrest and induction of apoptosis coupled with the down-regulation of NF-κB target genes that were involved in cell cycle progression (e.g. CCND1 and CDK6) and prevention of apoptosis such as Bcl-2, Bcl-xl, c-IAP2, and Survivin Respectively. Moreover, ATO-BIBR1532 significantly reduced the mRNA expression level of RELA, NFKB1, and several validated target genes of the NF-κB signaling pathway including NFKBIA, VEGFC, c-Myc, and hTERT. SIGNIFICANCE: The combination of ATO and BIBR1532 synergistically induced its anti-proliferative effect in breast cancer cells by targeting the two key cancer-related pathways, hTERT and NF-κB, and disrupting their feed-forward loop at the same time which result in the reduction of NF-κB transcriptional activity and subsequent down-regulation of its target genes.

Platensimycin-Encapsulated Liposomes or Micelles as Biosafe Nanoantibiotics Exhibited Strong Antibacterial Activities against Methicillin-Resistant Staphylococcus aureus Infection in Mice.

Platensimycin (PTM) is a promising natural product drug lead against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), while the clinical development was hampered by problems related to its poor solubility and pharmacokinetic properties. In this study, we used liposomes and micelles as carriers of PTM to prepare PTM nanoformulations for the treatment of MRSA infection in mice. PTM-loaded nanoparticles could effectively reduce residual bacteria in the MRSA-infected macrophage cell model, comparing to free PTM. More importantly, in vivo studies showed that encapsulation of PTM by liposomes or micelles effectively improved the pharmacokinetic properties of PTM in Sprague-Dawley rats and the survival rate of MRSA-infected C57BL/6J mice. Our study has thus suggested that the clinically used nanocarriers, such as liposome and micelle, might also be useful to improve the efficacy of other natural product drug leads to accelerate their in vivo evaluation and preclinical development.

An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia dissemination.

BACKGROUND: Current acute myeloid leukemia (AML) therapy fails to eliminate quiescent leukemic blasts in the bone marrow, leading to about 50% of patient relapse by increasing AML burden in the bone marrow, blood, and extramedullar sites. We developed a protein-based nanoparticle conjugated to the potent antimitotic agent Auristatin E that selectively targets AML blasts because of their CXCR4 receptor overexpression (CXCR4+) as compared to normal cells. The therapeutic rationale is based on the involvement of CXCR4 overexpression in leukemic blast homing and quiescence in the bone marrow, and the association of these leukemic stem cells with minimal residual disease, dissemination, chemotherapy resistance, and lower patient survival. METHODS: Monomethyl Auristatin E (MMAE) was conjugated with the CXCR4 targeted protein nanoparticle T22-GFP-H6 produced in E. coli. Nanoconjugate internalization and in vitro cell viability assays were performed in CXCR4+ AML cell lines to analyze the specific antineoplastic activity through the CXCR4 receptor. In addition, a disseminated AML animal model was used to evaluate the anticancer effect of T22-GFP-H6-Auristatin in immunosuppressed NSG mice (n = 10/group). U of Mann-Whitney test was used to consider if differences were significant between groups. RESULTS: T22-GFP-H6-Auristatin was capable to internalize and exert antineoplastic effects through the CXCR4 receptor in THP-1 and SKM-1 CXCR4+ AML cell lines. In addition, repeated administration of the T22-GFP-H6-Auristatin nanoconjugate (9 doses daily) achieves a potent antineoplastic activity by internalizing specifically in the leukemic cells (luminescent THP-1) to selectively eliminate them. This leads to reduced involvement of leukemic cells in the bone marrow, peripheral blood, liver, and spleen, while avoiding toxicity in normal tissues in a luminescent disseminated AML mouse model. CONCLUSIONS: A novel nanoconjugate for targeted drug delivery of Auristatin reduces significantly the acute myeloid leukemic cell burden in the bone marrow and blood and blocks its dissemination to extramedullar organs in a CXCR4+ AML model. This selective drug delivery approach validates CXCR4+ AML cells as a target for clinical therapy, not only promising to improve the control of leukemic dissemination but also dramatically reducing the severe toxicity of classical AML therapy.

THE EFFECT OF ANESTHETIC TIME AND CONCENTRATION ON BLOOD GASES, ACID-BASE STATUS, AND ELECTROLYTES IN KOI (CYPRINUS CARPIO) ANESTHETIZED WITH BUFFERED TRICAINE METHANESULFONATE (MS-222).

Anesthesia is commonly employed in aquatic medicine to facilitate physical exams, diagnostics, and surgical interventions. Tricaine methanesulfonate (MS-222) is the most commonly used anesthetic for fish and is currently the only anesthetic approved by the US Food and Drug Administration Center for Veterinary Medicine for food-producing fish. Despite the frequency of anesthetic procedures in fish, anesthetic monitoring remains rudimentary in many facilities. This study evaluated the impact on blood gases, acid-base balance, and electrolytes in koi (Cyprinus carpio) anesthetized at concentrations of 100 mg/L and 150 mg/L MS-222. Blood samples from 25 fish per treatment were collected at 5 and 20 min of anesthetic immersion. Forty-nine of 50 fish recovered uneventfully from anesthesia; one fish did not recover and was euthanatized. Results showed significant increases in partial pressure of carbon dioxide (pCO2) (P = 0.006) and hyperglycemia (P = <0.0001) with increasing anesthetic concentration and time under anesthesia and a significant decrease in partial pressure of oxygen (pO2) with increased anesthetic time (P = 0.021). There were several electrolyte changes observed with both increasing anesthetic time and concentration. All electrolytes except potassium remained within published reference ranges for koi, while potassium showed a significant decrease in concentration associated with anesthetic time and concentration. The results of this study indicate that MS-222 at 100 mg/L and 150 mg/L represent safe anesthetic concentrations for koi undergoing minimally invasive diagnostics; however, koi anesthetized with MS-222 at a concentration of 150 mg/L experienced more significant changes in blood gases, acid-base balance, and electrolyte concentrations.

Evaluation of Effective and Practical Euthanasia Methods for Larval African Clawed Frogs (Xenopus laevis).

Larval, or tadpole-stage Xenopus laevis frogs are a popular research model for developmental biology and disease studies. Existing euthanasia guidance documents offer recommendations for both eggs and adult stages, yet do not specifically address the larval stage. Data evaluating effective euthanasia methods for groups of X. laevis tadpoles would therefore be useful. The goal of the current study was to evaluate the efficacy of various immersion euthanasia procedures on tadpoles: tricaine methanesulfonate (MS222) at 6 g/L, eugenol at 800 µL/L and rapid chilling (2 to 4 °C). We also evaluated tadpoles at various developmental stages (NF stages 46, 47 and 49). Tadpoles (n = 70) were exposed to euthanasia solution for 15 min, and controls (n = 40) were placed in housing tank water for 15 min. All animals were then placed in recovery tanks containing housing tank water for 4 h to confirm irreversibility of each agent. Cessation of the heartbeat was assessed at the end of euthanasia solution exposure and at each hour thereafter. We found that immersion in a 6 g/L solution of MS222 resulted in 100% euthanasia of all larval stages tested. Conversely, eugenol produced variable euthanasia rates that were affected by both age group and batches of stock solutions. Rapid chilling was completely ineffective as a euthanasia method in our study. Based on our findings, we recommend MS222 as an effective and practical means of euthanizing large numbers of X. laevis tadpoles.

BIBR1532, a Selective Telomerase Inhibitor, Enhances Radiosensitivity of Non-Small Cell Lung Cancer Through Increasing Telomere Dysfunction and ATM/CHK1 Inhibition.

PURPOSE: Telomerase is reactivated in non-small cell lung cancer (NSCLC), and it increases cell resistance to irradiation through protecting damaged telomeres and enhancing DNA damage repair. We investigated the radiosensitizing effect of BIBR1532, a highly selective telomerase inhibitor, and its corresponding mechanism in NSCLC. METHODS AND MATERIALS: Cell proliferation, telomerase activity, and telomere dysfunction-induced foci were measured with CCK-8 assay, real-time fluorescent quantitative polymerase chain reaction, and immunofluorescence. The effect of BIBR1532 on the response of NSCLC cells to radiation was analyzed using clonogenic survival and xenograft tumor assays. Cell death and cell senescence induced by BIBR1532 or ionizing radiation (IR), or both, were detected with western blotting, flow cytometry, and senescence-association ß-galactosidase staining assay. RESULTS: We observed dose-dependent direct cytotoxicity of BIBR1532 at relatively high concentrations in NSCLC cells. Low concentrations of BIBR1532 did not appear toxic to NSCLC cells; however, they substantially increased the therapeutic efficacy of IR in vitro by enhancing IR-induced apoptosis, senescence, and mitotic catastrophe. Moreover, in a mouse xenograft model, BIBR1532 treatment synergized with IR at nontoxic dose levels promoted the antitumor efficacy of IR without toxicity to hematologic and internal organs. Mechanistically, lower concentrations of BIBR1532 effectively inhibited telomerase activity and increased IR-induced telomere dysfunction, resulting in disruption of chromosomal stability and inhibition of the ATM/CHK1 (ataxia-telangiectasia-mutated/Checkpoint kinase 1) pathway, which impaired DNA damage repair. CONCLUSIONS: Our findings demonstrate that disturbances in telomerase function by nontoxic dose levels of BIBR1532 effectively enhance the radiosensitivity of NSCLC cells. This finding provides a rationale for the clinical assessment of BIBR1532 as a radiosensitizer.

Evaluation of Platensimycin and Platensimycin-Inspired Thioether Analogues against Methicillin-Resistant Staphylococcus aureus in Topical and Systemic Infection Mouse Models.

Staphylococcus aureus is one of the most common pathogens causing hospital-acquired and community-acquired infections. Methicillin-resistant S. aureus (MRSA)-formed biofilms in wounds are difficult to treat with conventional antibiotics. By targeting FabB/FabF of bacterial fatty acid synthases, platensimycin (PTM) was discovered to act as a promising natural antibiotic against MRSA infections. In this study, PTM and its previously synthesized sulfur-Michael derivative PTM-2t could reduce over 95% biofilm formation by S. aureus ATCC 29213 when used at 2 µg/mL in vitro. Topical application of ointments containing PTM or PTM-2t (2 × 4 mg/day/mouse) was successfully used to treat MRSA infections in a BABL/c mouse burn wound model. As a potential prodrug lead, PTM-2t showed improved in vivo efficacy in a mouse peritonitis model compared with PTM. Our study suggests that PTM and its analogue may be used topically or locally to treat bacterial infections. In addition, the use of prodrug strategies might be instrumental to improve the poor pharmacokinetic properties of PTM.

Electroacupuncture plus on-demand gastrocaine for refractory functional dyspepsia: Pragmatic randomized trial.

BACKGROUND AND AIM: Treatment options for functional dyspepsia (FD) refractory to pharmacological treatments are limited but the effectiveness of electroacupuncture (EA) is uncertain. We assessed the effectiveness of EA combined with on-demand gastrocaine. METHODS: We conducted a single-center, assessor-blind, randomized parallel-group 2-arm trial on Helicobacter pylori negative FD patients of the postprandial distress syndrome subtype refractory to proton pump inhibitor, prokinetics, or H2 antagonists. Enrolled participants were block randomized in a 1:1 ratio, with concealed random sequence. The treatment and control groups both received on-demand gastrocaine for 12 weeks, but only those in treatment group were offered 20 sessions of EA over 10 weeks. The primary endpoint was the between-group difference in proportion of patients achieving adequate relief of symptoms at week 12. RESULTS: Of 132 participants randomly assigned to EA plus on-demand gastrocaine (n = 66) or on-demand gastrocaine alone (n = 66), 125 (94.7%) completed all follow-up at 12 weeks. The EA group had a compliance rate 97.7%. They had a significantly higher likelihood in achieving adequate symptom relief at 12 weeks, with a clinically relevant number needed to treat (NNT) value of 2.36 (95% CI: 1.74, 3.64). Among secondary outcomes, statistically and clinically significant improvements were observed among global symptom (NNT = 3.85 [95% CI: 2.63, 7.69]); postprandial fullness and early satiation (NNT = 5.00 [95% CI: 2.86, 25.00]); as well as epigastric pain, epigastric burning, and postprandial nausea (NNT = 4.17 [95% CI: 2.56, 11.11]). Adverse events were minimal and nonsignificant. CONCLUSION: For refractory FD, EA provides significant, clinically relevant symptom relief when added to on-demand gastrocaine (ChiCTR-IPC-15007109).

ENDOSCOPIC REMOVAL OF A FOREIGN BODY IN A MEXICAN AXOLOTL (AMBYSTOMA MEXICANUM) WITH THE USE OF MS222-INDUCED IMMOBILIZATION.

This communication briefly describes the use of tricaine methanesulfonate (MS222) to induce chemical restraint/general anesthesia of a Mexican axolotl (Ambystoma mexicanum) for the endoscopic retrieval of a gastric foreign body. There is very little published scientific literature concerning the anesthesia of Mexican axolotls. The anesthesia used in this case was an immersion bath of tricaine methanesulfonate where the concentration of tricaine methanesulfonate was gradually increased to 500 mg/L (ppm) over a 15-min period. A loss of righting reflex was observed within 3 min of attaining the final concentration of the anesthetic bath. The first voluntary movements following the transfer to a freshwater bath occurred within 7 min. The recovery was uneventful. Tricaine methanesulfonate in this case proved to be an effective anesthetic agent for a short, minimally invasive procedure.

Novel auristatin E-based albumin-binding prodrugs with superior anticancer efficacy in vivo compared to the parent compound.

Auristatins are a class of highly cytotoxic tubulin-disrupting peptides, which have shown limited therapeutic effect as free agents in clinical trials. In our continuing effort to develop acid-sensitive albumin-binding anticancer drugs exploiting circulating serum albumin as the drug carrier, we investigated the highly toxic drug payload auristatin E to assess whether the corresponding albumin-binding prodrugs were a viable option for achieving significant and concomitant tolerable antitumor activity. To achieve our goal, we developed a new aromatic maleimide-bearing linker (Sulf07) which enhanced both water solubility and stability of the prodrugs. In this study, we describe two auristatin E-based albumin-binding drugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, which were designed to release the active compound at the tumor site in a pH-dependent manner. These prodrugs incorporate an acid-sensitive hydrazone bond, formed by the reaction of a carbonyl-containing auristatin E derivative with the hydrazide group of the water-solubilizing maleimide-bearing linker Sulf07. A panel of patient- and cell-derived human tumor xenograft models (melanoma A375, ovarian carcinoma A2780, non-small-cell lung cancer LXFA737 and LXFE937, and head and neck squamous cell carcinomas) were screened with starting tumor volumes in the range of either 130-150 mm3 (small tumors) or 270-380 mm3 (large tumors). Both albumin-binding prodrugs showed compelling anticancer efficacy compared to the parent drug auristatin E, inducing statistically significant long-term partial and/or complete tumor regressions. AE-Keto-Sulf07 displayed very good antitumor response over a wide dose range, 3.0-6.5 mg/kg (5-8 injections, biweekly). AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg (8 injections, biweekly) or at 3.8 mg/kg (4 injections, weekly), but caused cumulative skin irritation due to scratching and biting. In contrast at its MTD, auristatin E (0.3 mg/kg, 8 injections, biweekly) was only marginally active. In summary, AE-Keto-Sulf07 and AE-Ester-Sulf07 are novel acid-sensitive albumin-binding prodrugs demonstrating tumor regressions in all of the evaluated human tumor xenograft models thus supporting the stratagem that albumin can be used as an effective drug carrier for the highly potent class of auristatins.

Use of tricaine methanesulfonate or propofol for immersion euthanasia of goldfish (Carassius auratus).

OBJECTIVE To substantiate current AVMA guidelines for immersion euthanasia of goldfish (Carassius auratus) with tricaine methanesulfonate (TMS), determine whether immersion in propofol at 5 times its immersion anesthesia concentration for 30 minutes is sufficient for euthanasia of goldfish, and quantify the duration of myocardial contraction following immersion of goldfish in TMS and decapitation. DESIGN Prospective clinical trial. ANIMALS 36 healthy, adult goldfish. PROCEDURES Goldfish were randomly assigned to be immersed in 1 of 6 test solution treatments (n = 6/treatment): TMS (500 mg/L) for 15 minutes followed by placement in anesthetic agent-free water (T15W), placement out of water (T15A), or decapitation (T15D); TMS (1,000 mg/L) for 15 minutes followed by placement in anesthetic agent-free water (T15XW); TMS (500 mg/L) for 30 minutes followed by placement in anesthetic agent-free water (T30W); or propofol (25 mg/L) for 30 minutes followed by placement in anesthetic agent-free water (P30W). Any fish that resumed operculation in group T15A was returned to anesthetic agent-free water. Times from onset of immersion to induction of anesthesia, cessation and resumption of operculation, and recovery (T15W, T15A, T15XW, T30W, P30W) or cessation of Doppler ultrasounds (T15D) were recorded. RESULTS Overall, 5 of 6, 6 of 6, 6 of 6, 6 of 6, and 5 of 6 fish survived in the T15W, T15A, T15XW, T30W, and P30W groups, respectively. Median time to cessation of Doppler ultrasounds in group T15D was 77.5 minutes (range, 30 to 240 minutes). CONCLUSIONS AND CLINICAL RELEVANCE Timed immersion in test solutions (TMS at 500 mg/L or 1,000 mg/L or propofol at 25 mg/L) resulted in death in only 7% (2/30) of immersed goldfish. Myocardial contractions continued for up to 4 hours in decapitated goldfish.

Comparison of Etomidate, Benzocaine, and MS222 Anesthesia with and without Subsequent Flunixin Meglumine Analgesia in African Clawed Frogs (Xenopus laevis).

Often few alternative anesthetics for exotic species are available, due to the small numbers of these animals used in research. In this study, we evaluated the depth and duration of anesthesia in Xenopus laevis after their immersion in 3 doses of etomidate (15, 22.5, and 30 mg/L) and in 3 doses of benzocaine (0.1%, 0.5%, and 1%) compared with the 'gold standard,' tricaine methanesulfonate (MS222; 2 g/L). We then chose an optimal dose for each alternative anesthetic according to induction time, duration of surgical plane, and time to complete recovery. The optimal etomidate and benzocaine doses (22.5 mg/L and 0.1%, respectively) as well as the MS222 dose were then used to achieve a surgical plane of anesthesia, with the addition of flunixin meglumine (25 or 50 mg/kg) administered in the dorsal lymph sac at the completion of mock oocyte harvest. Efficacy of the analgesic was assessed at 1, 3, 6, and 24 h postoperatively by using acetic acid testing (AAT). Histology of the liver, kidney, and tissues surrounding the dorsal lymph sac was performed at day 3, 14, and 28 in each group of animals. Mild to moderate myocyte degeneration and necrosis were present in tissues surrounding the dorsal lymph sac at both flunixin meglumine doses after etomidate and benzocaine anesthesia. In addition, the 50-mg/kg dose of flunixin meglumine resulted in the death of 5 of the 12 frogs within 24 h, despite an otherwise uneventful anesthetic recovery. In conclusion, benzocaine and etomidate offer alternative anesthetic regimens, according to typical requirements for an anesthetic event. Flunixin meglumine at the 25-mg/kg dose provided analgesic relief at the latest time point during etomidate dosage and at all time points during benzocaine dosage, but further characterization is warranted regarding long-term or repeated analgesic administration.

EV20-mediated delivery of cytotoxic auristatin MMAF exhibits potent therapeutic efficacy in cutaneous melanoma.

Cutaneous melanoma is one of the cancers with the fastest rising incidence and in its advanced metastatic form is a highly lethal disease. Despite the recent approval of several new drugs, the 5-year overall survival rate for advanced cutaneous melanoma is still below 20% and therefore, the development of novel treatments remains a primary need. Antibody-Drug Conjugates are an emerging novel class of anticancer agents, whose preclinical and clinical development has recently seen a remarkable increase in different tumors, including melanoma. Here, we have coupled the anti-HER-3 internalizing antibody EV20 to the cytotoxic drug monomethyl auristatin F (MMAF) to form a novel antibody-drug conjugate (EV20/MMAF). In a panel of human melanoma cell lines, this novel ADC shows a powerful, specific and target-dependent cell killing activity, independently of BRAF status. Efficacy studies demonstrated that a single administration of EV20/MMAF leads to a long-lasting tumor growth inhibition. Remarkably, the effect of this novel ADC was superior to the BRAF inhibitor vemurafenib in preventing kidney, liver and lung melanoma metastases. Overall, these results highlight EV20/MMAF as a novel ADC with promising therapeutic efficacy, warranting extensive pre-clinical evaluation in melanoma with high levels of HER-3 expression.