Mohamed O. Abdalla

Molecular cycloencapsulation augments solubility and improves therapeutic index of brominated noscapine in prostate cancer cells

We have previously shown that a novel microtubule-modulating noscapinoid, EM011 (9-Br-Nos), displays potent anticancer activity by inhibition of cellular proliferation and induction of apoptosis in prostate cancer cells and preclinical mice models. However, physicochemical and cellular barriers encumber the development of viable formulations for future clinical translation. To circumvent these limitations, we have synthesized EM011-cyclodextrin inclusion complexes to improve solubility and enhance therapeutic index of EM011. Phase solubility analysis indicated that EM011 formed a 1:1 stoichiometric complex with β-CD and methyl-β-CD, with a stability constant (K(c)) of 2.42 × 10(-3) M and 4.85 × 10(-3) M, respectively. Fourier transform infrared spectroscopy suggested the penetrance of either a O-CH(2) or OCH(3)-C(6)H(4)-OCH(3) moiety of EM011 in the β-CD or methyl-β-CD cavity. In addition, multifarious techniques, namely, differential scanning calorimetry, powder X-ray diffraction, scanning electron microscopy, NMR spectroscopy, and computational studies validated the cage complex of EM011 with β-CD and methyl-β-CD. Moreover, rotating frame overhauser enhancement spectroscopy showed that the H(a) proton of the OCH(3)-C(6)H(4)-OCH(3) moiety was in close proximity with H3 proton of the β-CD or methyl-β-CD cavity. Furthermore, we found that the solubility of EM011 in phosphate buffer saline (pH 7.4) was enhanced by ~11 fold and ~21 fold upon complexation with β-CD and methyl-β-CD, respectively. The enhanced dissolution of the drug CD-complexes in aqueous phase remarkably decreased their IC(50) to 28.5 μM (9-Br-Nos-β-CD) and 12.5 μM (9-Br-Nos-methyl-β-CD) in PC-3 cells compared to free EM011 (~200 μM). This is the first report to demonstrate the novel construction of cylcodextrin-based nanosupramolecular vehicles for enhanced delivery of EM011 that warrants in vivo evaluation for the superior management of prostate cancer.

Cholecystokinin-33 is more effective than cholecystokinin-8 in inhibiting food intake and in stimulating the myenteric plexus and dorsal vagal complex

We compared the abilities of cholecystokinin-33 (CCK-33) and CCK-8 to reduce food intake and to activate feeding-related areas of the nervous system. (1) Overnight food-deprived rats were presented with a 10% sucrose solution, and intake was measured at 5-min intervals throughout a 90-min test beginning immediately after intraperitoneal injections of 1, 3, or 5 nMol/kg of CCK-33, CCK-8, or the vehicle control. In the initial 20 min (first meal), both peptides were equally effective, producing large reductions of food intake. Thereafter, however, CCK-33 was more effective than CCK-8, producing much more sustained reductions. Overall, both peptides reduced total food intake, but CCK-33 was more effective than CCK-8. (2) Possible roles for the myenteric plexus of the duodenum and the dorsal vagal complex (DVC) of the brainstem in the differential satiety effects of CCK-33 and CCK-8 were examined by quantifying CCK-33- and CCK-8-stimulated Fos-like immunoreactivity (Fos-LI) in each site. Consistent with the greater ability of CCK-33 to produce sustained inhibitions of food intake, CCK-33 produced more Fos-LI than CCK-8 in nearly every section of the sampled sites. The results demonstrate: (1) Different forms of CCK have different efficacies in reducing food intake; (2) CCK-33 produces a much more prolonged satiety action than CCK-8; and (3) the myenteric plexus and DVC may play roles in these differential satiety actions.

Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex.

The enteric nervous system (ENS: myenteric and submucosal plexuses) of the gastrointestinal tract may have a role in the reduction of food intake by cholecystokinin (CCK). Exogenous cholecystokinin-8 (CCK-8) activates the myenteric plexus and the feeding control areas of the dorsal vagal complex (DVC) of the brainstem. An increasing number of reports, however, have shown that CCK-58 is the sole or the major circulating form of CCK in rat, human and dog, and that it is qualitatively different from CCK-8 in evoking various gastrointestinal physiological responses (e.g., contraction of the gallbladder and exocrine pancreatic secretion). In the current report, we compared the abilities of exogenous CCK-58 to activate the myenteric plexus and the dorsal vagal complex with those of exogenous CCK-8 by quantifying Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). We report that CCK-58 (1, 3, and 5 nmol/kg) increased Fos-LI in the myenteric plexus (p<0.001) and in the DVC (p<0.001) compared to the saline vehicle. The highest dose of CCK-58 increased Fos-LI more than an equimolar dose of CCK-8 in the myenteric plexus and the area postrema. Thus, CCK-8 and CCK-58 produce the same qualitative pattern of activation of central and peripheral neurons, but do not provoke identical quantitative patterns at higher doses. The different patterns produced by the two peptides at higher doses, in areas open to the circulation (myenteric plexus and area postrema) may reflect endocrine actions not observed at lower doses.

Chemotherapy of Prostate Cancer by Targeted Nanoparticles Trackable by Magnetic Resonance Imaging

Prostate cancer (CaP) is the commonest diagnosed malignancy and the second main cause of cancer mortality in males in the United States. Thus, there is an urgent need to develop novel drug delivery systems to improve the chemotherapy option for CaP patients. The goal of this paper is to describe novel moleculary guided nanoscale drug delivery system with dual functionality for treatment and MR imaging of CaP. We describe the synthesis of iron oxide nanoparticles (IONPs) which are then coated with carboxyl-ended amphiphilic polymer. We present the protocol for tethering of the CaP targeting protein, human amino terminal fragment (hATF) to the terminal carboxyls of the IONPs. We describe the drug loading and release and the methods for measuring of the internalization of the hATF-guided IONPs into CaP cells. We also describe the methods for usages of IONPs are MR imaging contrast agent and successful targeted drug carriers.

Double-receptor-targeting multifunctional iron oxide nanoparticles drug delivery system for the treatment and imaging of prostate cancer

As an alternative therapeutic treatment to reduce or eliminate the current side effects associated with advanced prostate cancer (PCa) chemotherapy, a multifunctional double-receptor-targeting iron oxide nanoparticles (IONPs) (luteinizing hormone-releasing hormone receptor [LHRH-R] peptide- and urokinase-type plasminogen activator receptor [uPAR] peptide-targeted iron oxide nanoparticles, LHRH-AE105-IONPs) drug delivery system was developed. Two tumor-targeting peptides guided this double-receptor-targeting nanoscale drug delivery system. These peptides targeted the LHRH-R and the uPAR on PCa cells. Dynamic light scattering showed an increase in the hydrodynamic size of the LHRH-AE105-IONPs in comparison to the non-targeted iron oxide nanoparticles (NT-IONPs). Surface analysis showed that there was a decrease in the zeta potential values for drug-loaded LHRH-AE105-IONPs compared to the NT-IONPs. Prussian blue staining demonstrated that the LHRH-AE105-IONPs were internalized efficiently by the human PCa cell line, PC-3. In vitro, magnetic resonance imaging (MRI) results confirmed the preferential binding and accumulation of LHRH-AE105-IONPs in PC-3 cells compared to normal prostate epithelial cells (RC77N/E). The results also showed that LHRH-AE105-IONPs significantly maintained T2 MRI contrast effects and reduced T2 values upon internalization by PC-3 cells. These paclitaxel-loaded double-receptor-targeting IONPs also showed an approximately twofold reduction in PC-3 cell viability compared to NT-IONPs.

Abstract 2170: Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radiotherapies, targeted drug delivery is now gaining the focus of cancer researchers. The key for success of any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Two over-expressed such receptors, which can be targeted to the cell surface of PCa cells are: Gonadotropin Releasing Hormone Receptor (GnRH-R), and Prostate Specific Membrane Antigen (PSMA). The analysis done from molecular dynamics simulation data gave an important information about each interaction; such as RMSD, The number of contacts between receptor and peptide within a cutoff distance of 5 A and the binding free energies, ΔGGBSA of the interactions. In all systems, the back bone RMSD deviation of the receptor was calculated using the last 10 ns of the sampling process and averaged over all trajectories. The RMSD data deviated between 1.22 - 2.51 A. The free binding energies or ΔGGBSA was calculated from the last 10 ns of the sampling process. The QH1 system has the more negative ΔGGBSA -36.15 kcal mol-1 and the QH4 system has the least negative ΔGGBSA of -18.4 kcal mol-1 among the eight systems of GnRH-R. QH2 system exhibits a highly interactive hydrogen bond (bond distance is 2.65 A) between 134th position of the receptor amino acid sequence GLU and 5th position of the qh2 peptide9s TYR. The ΔGGBSA is for the qh2 system is – 34.92 kcal mol-1. In the PSMA and its targeting peptide interactions, T2IA has the least negative ΔGGBSA of -47.37 kcal mol-1. A salt bridge was observed between the 10th position of the T2IA peptide sequence ARG and the 328 position of the 3RBU receptor9s GLU. Based on our molecular modeling studies, we have determined the best targeting peptides among all the newly designed peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated surface plasmon resonance (SPR) in near future. Citation Format: Ahmad Salam, Vincent Hembrick, Jesse Jaynes, Timothy Turner, Mohamed Abdalla. Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2170.

Abstract 3675: Double receptor targeting multifunctional iron oxide nanoparticles drug delivery system for the treatment and imaging of prostate cancer

Purpose: As an alternative to the drawbacks of current advanced prostate cancer chemotherapy, we propose a multifunctional double targeting drug delivery system that utilizes the combination of two cancer-targeting peptides: a modified luteinizing hormone releasing hormone (LHRH), the ligand for luteinizing hormone releasing hormone receptor (LHRH-R), and AE105, the ligand for urokinase type plasminogen activation receptor (uPAR). Both ligands are fused to amphiphilic polymer coated iron oxide nanoparticles (IONPs) and loaded with the anticancer drug Paclitaxel (PTX) as the payload. Materials and Methods: Targeted IONPs were characterized by both gel electrophoresis and dynamic light scattering (DLS). PTX was absorbed on the IONPs. Drug loading and release characteristics were conducted by HPLC. The binding specificity and the internalization efficiency of the targeted IONPs were both examined by Prussian blue staining and magnetic resonance imaging (MRI). Cytotoxicity of the targeted IONPs was evaluated by MTT assay. Results: Characterization of targeted IONPs by gel electrophoresis confirmed the successful attachment of the peptides to IONPs. Conjugation of peptides to carboxylic groups of polymer coating on IONPs resulted in an increase in the average hydrodynamic size of targeted IONPs (16.34 nm) as compared to non-targeted IONPs (12.51 nm), as well as a decrease of zeta potential from -70.43 mV to -58.06 mV, respectively. Prussian blue staining demonstrated that both, LHRH and AE105 conjugated IONPs were internalized efficiently by cells of the human prostate cancer cell line, PC3. In vitro MRI results showed that double-targeted IONPs significantly maintained T2 MRI contrast effect and reduction of T2 values upon internalization by PC3 cells. MRI imaging confirmed the preferential binding and accumulation of double-targeted IONPs in PC3 cells when compared to normal prostate epithelial cells (RC77N/E). PTX loaded double-targeted IONPs were stable at physiological pH and efficiently released around pH 4, the optimum pH inside the tumor cells. PTX loaded double-targeted IONPs showed an approximately 2-fold reduction in PC3 cell viability when compared to non-targeted IONPs. In addition, the percentage of cell death resulting from the PTX loaded, double-targeted IONPs was very similar to the percentage of cell death attributed to 100 ng/ml of free PTX, thereby demonstrating that their capability of reducing drug concentration. Conclusions: Our results indicate that we have developed a LHRH-R and uPAR targeted IONPs drug delivery system that potentially provides a MRI tractable delivery of cancer therapeutics such as PTX to prostate cancer cells. Therefore, our optimized double-targeted IONPs drug delivery system has the potential to significantly improve the health outcomes and quality of life for cancer patients as a novel type of targeted nanomedicine therapy. Citation Format: Md Shakir U. Ahmed, Mohamed O. Abdalla, Clayton Yates, Jesse Jaynes, Timothy Turner. Double receptor targeting multifunctional iron oxide nanoparticles drug delivery system for the treatment and imaging of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3675. doi:10.1158/1538-7445.AM2015-3675

Abstract 3694: Molecular modeling studies of novel receptor targeted peptides in the treatment of prostate cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Purpose: Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radio-therapies, targeted drug delivery is now gaining focus of cancer researchers. The key for success for any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Four over-expressed receptors which can be targeted on the cell surface of PCa cells are: (GnRH), (EGFR), (PSMA), and (uPAR). Thus, we propose to study the binding affinity of tweleve novel designed targeting peptides (three peptides per receptor) for these over-expressed receptors. Initially, we will study the binding affinity using a computerized molecular modeling program. We will corroborate our modeling data by determining the dissociation constants of the peptide-receptor interaction using sensitive experimental tools such as miscroscale thermophoresis. Methods: The newly designed peptides were generated in pdb (protein database) format and the known pdb files of the receptors were collected from protein data bank. Cluspro 2.0, developed by Boston University, is a protein docking server with a molecular modeling program that was used to calculate the binding energies of these peptides to their corresponding receptors. The data obtained was analyzed using Pymol. Results: The molecular docking studies showed that the GnRH tarteting peptide QH10, uPAR targeting peptide CV13, EGFR targeting peptide RL10, and PSMA targeting peptide TH12 have the lowest mean binding energies for their respective top three binding conformations. The calculatd binding energies for the aftermentioed peptides-receptors combination are: -1285 KCal/M, -1029 KCal/M, -1019 KCal/M and -1037 KCal/M, respectively. There results suggested a very high interaction for these newly designed peptides and their corroponding receptors as the typical calculated binding energy for an antigen-antibody interaction is -700 KCal/M. Conclusion: Based on our molecular docking studies, we have determined the best targeting peptides among all the newly desiged peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated using microscale thermophoresis. This work is supported by grant funding from NIH/RCMI G12MD00758 (T. Turner) and NIH/NCI U54 CA118623 (T. Turner & M. Abdalla). Note: This abstract was not presented at the meeting. Citation Format: Mohamed O. Abdalla, Ahmad Bin Salam, Vincent Hembrick, Manikanthan Bhavaraju, Clayton Yates, Jesse Jaynes, Timothy Turner. Molecular modeling studies of novel receptor targeted peptides in the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2015-3694

Abstract 4480: Double targeting nanoscale drug delivery system for treatment and imaging of metastatic solid cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: As an alternative to the drawbacks of current advanced cancer treatments such as conventional chemotherapy, we propose a multifunctional double targeting drug delivery system that utilizes the combination of cancer-targeting peptides fused to amphiphilic polymer coated iron oxide nanoparticles (IONPs) and loaded with suitable anticancer drugs as the payload. Methods: PC3 human prostate cancer cell line will be used initially to test the efficiency of the proposed drug delivery system. Our target sites of choice are the luteinizing hormone releasing hormone receptor (LHRH-R) and the urokinase-type plasminogen activator receptor (uPAR), and docetaxel was selected as the payload. A modified LHRH (ligand for LHRH-R) and AE105 (ligand for uPAR) were conjugated to polymer coated IONPs according to the manufacturers protocol. Results: Conjugated IONPs were characterized by gel electrophoresis and Dynamic Light Scattering (DLS). IONPs showed expected narrow size distribution after conjugation with peptide ligand. The average hydrodynamic size of non targeted IONPs (36.84 nm) was increased upon conjugation of double peptide to their surface (44.06 nm). Conjugation of peptides to carboxylic groups of polymer coating on IONPs resulted in a decrease of zeta potential from -70.43 mV to -58.06 mV. Prussian blue staining demonstrated that LHRH and AE105 conjugated IONPs were internalized efficiently by the PC3 cells. The drug loading and release capability of conjugated IONPs will be evaluated by HPLC. In addition the internalization of conjugated IONPs will also be examined utilizing Magnetic Resonance Imaging (MRI), and tumor eradication will be determined by MTT assay. Conclusions: We expect a significant enhancement in the binding efficiency of the LHRH-IONPs-AE105 to the prostate cancer cells, increased efficiency in tumor eradication, and better imaging and monitoring capabilities because IONPs can be visualized by MRI during molecular imaging of the prostate cancer cells. Therefore, we believe the optimization of the proposed system will enhance targeted nanomedicine and significantly improve the health outcomes and quality of life for cancer patients because of the following reasons: 1) its ability to deliver anticancer drugs specifically to cancer cells while sparing the surrounding normal tissues, and 2) the capability of in situ monitoring of the therapeutics. Citation Format: Md Shakir U. Ahmed, Mohamed O. Abdalla, Timothy Turner. Double targeting nanoscale drug delivery system for treatment and imaging of metastatic solid cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4480. doi:10.1158/1538-7445.AM2014-4480