Keren Sasson

Angiotensin 1-7 as Means to Prevent the Metabolic Syndrome: Lessons From the Fructose-Fed Rat Model

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Turning low-molecular-weight drugs into prolonged acting prodrugs by reversible pegylation: a study with gentamicin.

Pegylation is a powerful technology to prolong the action of proteins in vivo, but it is impractical for low-molecular-weight (LMW) drugs, which are usually inactivated upon such modification. Here, we have applied a recently developed strategy of reversible pegylation to gentamicin, a LMW antibiotic. Variable length polyethyleneglycol (PEG-SH) chains were covalently linked to gentamicin using two heterobifunctional agents, each containing a spontaneously hydrolyzable bond. The inactive derivatives regained full antibacterial potency upon incubation under physiological conditions in vitro, and following systemic administration to rats, they released native active gentamicin with half-lives 7- to 15-fold greater than those of systemically administered nonderivatized gentamicin. In conclusion, reversibly pegylated prodrug derivatives of gentamicin were found to be capable of releasing gentamicin for prolonged periods in vivo. Most importantly, the major drawback of conventional pegylation, namely, the loss of pharmacological potency following irreversible derivatization, has been overcome.

Enhanced in Vivo Efficacy of a Type I Interferon Superagonist with Extended Plasma Half-life in a Mouse Model of Multiple Sclerosis

Background: IFNβ constitutes an approved drug to treat multiple sclerosis (MS), but it has limited efficacy. Results: A modified human IFN variant, which exhibits both superagonist properties and 10-fold increased lifespan, outperforms IFNβ in an animal MS model. Conclusion: This drug candidate has potential to supersede IFNβ for the treatment of MS. Significance: Protein engineering allows development of more effective drugs to treat autoimmune diseases. IFNβ is a common therapeutic option to treat multiple sclerosis. It is unique among the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNSα8) that is built on the backbone of a low affinity IFNα but modified to exhibit higher receptor affinity than even for IFNβ. Here, YNSα8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain comprising proline, alanine, and serine (PAS) to prolong its plasma half-life via “PASylation.” PAS-YNSα8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors, notably without any detectable loss in biological potency or bioavailability. This long-lived superagonist conferred significantly improved protection from MOG35–55-induced experimental autoimmune encephalomyelitis compared with IFNβ, despite being injected with a 4-fold less frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS measurements showing a decrease of CD11b+/CD45hi myeloid lineage cells detectable in the CNS, as well as a decrease in IBA+ cells in spinal cord sections determined by immunohistochemistry for PAS-YNSα8-treated animals. Importantly, PAS-YNSα8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. A striking correlation between increased levels of CD274 (PD-L1) transcripts from spleen-derived CD4+ cells and improved clinical response to autoimmune encephalomyelitis was observed, indicating that, at least in this mouse model of multiple sclerosis, CD274 may serve as a biomarker to predict the effectiveness of IFN therapy to treat this complex disease.

Engineering prolonged-acting prodrugs employing an albumin-binding probe that undergoes slow hydrolysis at physiological conditions

Here we describe the design and application of OSu-FMS-MAL-S-(CH(2))(15)-COOH, an agent that associates with albumin while linked to a peptide or a protein with sufficient affinity (Ka=2 to 2.6 x 10(5)M(-1)) to protract the action of short- lived peptides and proteins in vivo. Under physiological conditions this probe undergoes spontaneous hydrolysis with the concomitant reactivation of inactive conjugates. Intravenously administered (125)I-labeled-Insulin-FMS-MAL-S-(CH(2))(15)-COOH to rats shows half-life of 17+/-2h, exceeding 5.2 times that obtained with intravenously administered (125)I-labeled Insulin. In mice this derivative facilitates glucose-lowering effect over a period of 24h, yielding AUC five times greater than that obtained by a similar dose of insulin-detemir. Similarly, subcutaneous administration of Exendin-4-FMS-MAL-S-(CH(2))(15)-COOH into mice facilitated prolonged and stable reduction in glucose level, yielding a t(1/2) value of 28+/-2h, exceeding the effect of exendin-4 4.7 folds. The inactive derivative gentamicin-FMS-MAL-S-(CH(2))(15)-COOH regained its full antibacterial potency upon incubation at physiological conditions yielding a t(1/2) value of 7.1+/-0.2h. In conclusion, the albumin-binding probe we introduced enables to prolong the action of any amino containing molecule in vivo, without the drawback of inactivation that often occurs upon such derivatization.

Delivery of Neuropeptides from the Periphery to the Brain: Studies with Enkephalin

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Peptide derived from HIV-1 TAT protein, destabilizes a monolayer of endothelial cells in an in vitro model of the blood-brain barrier, and allows permeation of high molecular weight proteins.

Background: An in vitro model for studying BBB opening and entry of impermeable substances was constructed. Results: A brain capillary endothelial cell monolayer was disrupted and opened to the penetration of impermeable therapeutic agents by C-TAT peptides. Conclusion: Experimental conditions enabling the blood to brain entry of impermeant therapeutic agents were established. Significance: Approaches toward overcoming states of major brain disorders were initiated. Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.

Newly designed modifier prolongs the action of short-lived peptides and proteins by allowing their binding to serum albumin.

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Palliative treatment of obstructive esophageal cancer by vascular targeted photodynamic therapy (VTP): Preclinical study in orthotopic rat model.

59 Background: Vascular targeted photodynamic therapy (VTP) based on local sensitization of circulating WST11 (Pd-bacteriochlorophyll), enables effective focal ablation of solid tumors. WST11 clears from the circulation with t1/2 ~40 min (in humans) and does not accumulate in tissues which assures high level of safety and minimal adverse effects. Here we evaluate WST11-VTP for the treatment of obstructive esophageal cancer aiming at developing clinically relevant protocol. Methods: Using endoscopy guided procedure rat esophageal cancer cells (JA) were implanted in the mid esophagus. Following intravenous infusion of WST11, established tumors were illuminated by cylindrical fiber using the same endoscope sleeve. Safety and efficacy were evaluated by monitoring animal weight shift and histology of treated esophageal tissues. Treated animals included control (light/no WST11 or WST11/no light), WST11-VTP and WST11-VTP following treatment with bevacizumab at day -3. Results: WST11-VTP on esophageal tissues res...

Abstract B012: Immunomodulated VTP enables cure of metastatic prostate and breast cancers in animal models

Vascular-targeted photodynamic therapy with WST11 (VTP) enables safe and efficient non-thermal ablation of solid tumors as demonstrated by recent phase II and III clinical trials with localized prostate cancer. Studies in animal models in our labs revealed that VTP of immunogenic tumors elicits innate/adaptive immune responses and over 80% cures with consequent long-lasting anti-tumor protection. Here we show that local VTP combined with immune modulation in rodents, bearing weakly immunogenic prostate and triple negative breast tumors, results in eradication of remote metastases and animal cure. Methodology: 4T1-luc cells were subcutaneously grafted at the hind leg of Balb/C mice. Colony forming assay confirmed tumor cell spreading to the lungs already by day 7 post grafting when local VTP was delivered to the primary tumor. The study comprised: (i) animals treated by VTP alone or (ii) following 50mg/kg cyclophosphamide (CTX) on day 4 post grafting, or (iii) VTP combined with anti-CTLA4 antibodies administered on days 1, 4, 7 and 10 post VTP according to standard protocol. Appropriate control groups using immunomodulatory agents alone or untreated tumors were utilized. Cure was defined as absence of detectable primary or metastatic tumor by necropsy at 90 days. Mat-Lu-luc prostate cancer cells were grafted orthotopically in Copenhagen rats. Surgical removal of tumor bearing prostate and follow up of disease progression demonstrated that on day 10 post grafting, the day of VTP, metastatic spread has already been initiated in 57% of animals, eventually developing abdominal and visceral metastases. Treatment groups comprised (i) animals treated by VTP with or (ii) without CTX pre-treatment followed by prostate removal three days after VTP or (iii) surgery only. Results: Treatment of primary 4T1-luc tumors with VTP resulted in a complete primary tumor ablation in 50% of treated animals. Less then 10% of animals treated with VTP alone were cured with the rest developing lung metastases, while combination with CTX resulted in 30% cures. CTX induced reduction of total cell counts in tumor draining lymph nodes (LN) and spleens on VTP day followed by cell expansion over the following four days. Significant CTX-mediated reduction of Foxp3+ cells accompanied by increase in CD8+ cells during this period could be responsible for observed anti-metastatic effect of the combined treatment. Moreover, no significant change in total CD4+ cells observed at the same time suggests expansion of T helper cell subsets with concomitant suppression of regulatory population. Anti-CTLA-4 treatment, although significantly improved primary response rates after sub-optimal VTP (56% vs 14% in combined group vs VTP), did not prevent metastases suggesting additional escape mechanisms. VTP of orthotopic prostate tumors followed by whole gland removal resulted in 45% metastatic rate vs 57% in group treated by surgery alone on the same day. Pre-treatment with 50mg/kg CTX three days prior to VTP reduced metastatic rate to 7%. These findings suggest VTP-induced immune response can be augmented by prior immune modulation. Conclusions: Our results demonstrate that immunomodulation combined with local VTP enables systemic anti-tumor effects with impact on the development of early metastatic disease in the setting of poorly immunogenic tumors. The choice of different immune modulatory agents and timing of treatment appears to play a critical role in the treatment outcome. Further studies are in progress to evaluate combination of WST11-VTP with more than one immunomodulating agent to further characterize localized and systemic response and augment treatment success. These studies serve as pre-clinical support for clinical trials, exploiting the immunogenic effects of VTP for tumor ablation, being developed in collaboration with Memorial Sloan Kettering Cancer Center. Note: This abstract was not presented at the conference. Citation Format: Dina Preise, Natasha Kudinova, Uri Lindner, Keren Sasson, Ronny Uzana, Jonathan Coleman, Yoram Salomon, Avigdor Scherz. Immunomodulated VTP enables cure of metastatic prostate and breast cancers in animal models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B012.