- Doxorubicin Delivery into Tumor Cells by Stable Cavitation without Contrast Agents. [Journal Article]
- MPMol Pharm 2017 Jan 20
- Doxorubicin, alone or in combination with other anticancer agents, is one of the most widely used chemotherapeutic agents and is administered in a wide range of cancers. However, the use of doxorubic...
Doxorubicin, alone or in combination with other anticancer agents, is one of the most widely used chemotherapeutic agents and is administered in a wide range of cancers. However, the use of doxorubicin is limited due to its potential serious adverse reactions. Previous studies have established the ability of high intensity focused ultrasound (HIFU) in combination with various contrast agents to increase intracellular doxorubicin delivery in a targeted and noninvasive manner. In this study, we developed a new sonoporation device generating and monitoring acoustic cavitation bubbles without any addition of contrast agents. The device was used to potentiate the delivery of active doxorubicin into both adherent and suspended cell lines. Combining doxorubicin with ultrasound resulted in a significant enhancement of doxorubicin intracellular delivery and a decrease in cell viability at 48 and 72 h, in comparison to doxorubicin alone. More importantly and unlike previous investigations, our procedure does not require the addition of contrast agents to generate acoustic cavitation and to achieve high levels of doxorubicin delivery. The successful translation of this approach for an in vivo application may allow a significant reduction in the dosage and the adverse effects of doxorubicin therapy in patients.
- Food Protein-based Core-Shell Nanocarriers for Oral Drug Delivery: Effect of Shell Composition on in-vitro and in-vivo Functional Performance of Zein Nanocarriers. [Journal Article]
- MPMol Pharm 2017 Jan 19
- The study was aimed at systematically investigating the influence of shell composition on the particle size, stability, release, cell uptake, permeability and in-vivo gastrointestinal distribution of...
The study was aimed at systematically investigating the influence of shell composition on the particle size, stability, release, cell uptake, permeability and in-vivo gastrointestinal distribution of food protein-based nanocarriers for oral delivery applications. Three different core-shell nanocarriers were prepared using food-grade biopolymers including zein-casein (ZC) nanoparticles, zein-lactoferrin (ZLF) nanoparticles and zein-PEG (ZPEG) micelles. Nile red was used as a model hydrophobic dye for in-vitro studies. The nanocarriers had negative, positive and neutral charge respectively. All the three nanocarriers had a particle size of less than 200 nm and a low polydispersity index. The nanoparticles were stable at gastrointestinal pH (2-9) and ionic strength (10-200mM). The nanocarriers sustained the release of Nile red in simulated gastric and intestinal fluids. ZC nanoparticles showed the slowest release followed by ZLF nanoparticles and ZPEG micelles. The nanocarriers were taken up by endocytosis in Caco-2 cells. ZPEG micelles showed the highest cell uptake and transepithelial permeability followed by ZLF and ZC nanoparticles. ZPEG micelles also showed P-gp inhibitory activity. All the three nanocarriers showed bioadhesive properties. Cy 5.5, a near IR dye was used to study the in-vivo biodistribution of the nanocarriers. The nanocarriers showed longer retention in the rat gastrointestinal tract compared to the free dye. Among the three formulations, ZC nanoparticles was retained the longest in the rat gastrointestinal tract (≥24 hours). Overall, the outcomes from this study demonstrate the structure-function relationship of core-shell protein nanocarriers. The findings from this study can be used to develop food protein based oral drug delivery systems with specific functional attributes.
- A gemcitabine based peptide conjugate with improved metabolic properties and dual mode of efficacy. [Journal Article]
- MPMol Pharm 2017 Jan 18
- Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabol...
Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide, and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current manuscript provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC-MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake and release of gemcitabine and quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an in vitro colony forming assay together with in vivo whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine's bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single non-toxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution and safety, while also agonistically ablating testosterone levels.
- Computational models of the gastrointestinal environment 1. The effect of digestion on the phase behaviour of intestinal fluids. [Journal Article]
- MPMol Pharm 2017 Jan 18
- Improved models of the gastrointestinal environment have great potential to assist the complex process of drug formulation. Molecular dynamics (MD) is a powerful method for investigating phase behavi...
Improved models of the gastrointestinal environment have great potential to assist the complex process of drug formulation. Molecular dynamics (MD) is a powerful method for investigating phase behaviour at a molecular level. In this study we use multiple MD simulations to calculate phase diagrams for bile before and after digestion. In these computational models, undigested bile is represented by mixtures of palmitoyl-oleoylphosphatidylcholine (POPC), sodium glycodeoxycholate (GDX) and water. Digested bile is modelled using a 1:1 mixture of oleic acid and palmitoylphosphatidylcholine (lyso-phosphatidylcholine, LPC), GDX and water. The computational phase diagrams of undigested and digested bile are compared and we describe the typical in-termolecular interactions that occur between phospholipids and bile salts. The diffusion coefficients measured from MD simulation are compared to experimental diffusion data measured by DOSY-NMR, where we observe good qualitative agreement. In an addi-tional set of simulations, the effect of different ionization states of oleic acid on micelle formation is investigated.
- Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy. [Journal Article]
- MPMol Pharm 2017 Jan 17
- Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the ration...
Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.
- Radiolabeled R954 derivatives for imaging bradykinin B1 receptor expression with positron emission tomography. [Journal Article]
- MPMol Pharm 2017 Jan 17
- Peptide receptors have emerged as promising targets for diagnosis and therapy. The aberrant overexpression of these receptors in different cancer subtypes allows for the adoption of new treatment str...
Peptide receptors have emerged as promising targets for diagnosis and therapy. The aberrant overexpression of these receptors in different cancer subtypes allows for the adoption of new treatment strategies that complement conventional chemotherapies. Bradykinin B1 receptor (B1R) is a G protein-coupled receptor that is overexpressed in many cancers, with limited expression in healthy tissues. Previously, we developed 68Ga- and 18F-labeled derivatives of B1R antagonist peptides B9858 and B9958, and successfully targeted B1R-expressing tumor xenografts in vivo. R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-D-2-Nal-Ile), a potent B1R antagonist, is reportedly more stable than B9858 against peptidase degradation. We evaluated two radiolabeled derivatives of R954 (68Ga-HTK01083 and 18F-HTK01146) for B1R PET imaging. Peptides were assembled on were synthesized via solid phase strategy. Non-radioactive standards were obtain by reacting GaCl3 with DOTA-dPEG2-R954, and by clicking N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate with azidoacetyl-dPEG2-R954. Binding affinity for B1R was determined by an in vitro competition binding assay. 68Ga-HTK01083 was obtained by incubating DOTA-dPEG2-R954 with 68GaCl3 under acidic conditions, while 18F-HTK01146 was prepared via an 18F-19F isotope exchange reaction. Biodistribution and imaging studies were conducted at 1 h post-injection (p.i.) in mice inoculated with B1R-expressing (B1R+) and B1R-nonexpressing (B1R-) cells. HTK01083 and HTK01146 bound B1R with good affinity (Ki = 30.5 and 24.8 nM, respectively). 68Ga/18F-labeled R954 were obtained on average in ≥ 10% decay-corrected radiochemical yield with > 99% radiochemical purity and ≥ 52 GBq/μmol specific activity. For both tracers, clearance was predominantly renal with minimal involvement of the hepatobiliary system. For PET images, B1R+ tumors, kidneys and bladder were visible. At 1 h p.i., uptake in B1R+ tumor was comparable between 68Ga-HTK01083 (8.46 ± 1.44 %ID/g) and 18F-HTK01146 (9.25 ± 0.69 %ID/g). B1R+ tumor-to-blood and B1R+ tumor-to-muscle ratios were 6.32 ± 1.44 and 20.7 ± 3.58 for 68Ga-HTK01083, and 7.24 ± 2.56 and 19.5 ± 4.29 for 18F-HTK01146. Our results indicate R954 is a good lead sequence for optimization of B1R tracers for cancer imaging.
- Backbone Degradable HPMA Copolymer Conjugates with Gemcitabine and Paclitaxel: Impact of Molecular Weight on Activity toward Human Ovarian Carcinoma Xenografts. [Journal Article]
- MPMol Pharm 2017 Jan 17
- Degradable diblock and multiblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer - gemcitabine (GEM) and - paclitaxel (PTX) conjugates were synthesized by RAFT copolymerization followed by click ...
Degradable diblock and multiblock N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer - gemcitabine (GEM) and - paclitaxel (PTX) conjugates were synthesized by RAFT copolymerization followed by click reaction for preclinical investigation. The aim was to validate the hypothesis that long-circulating conjugates are needed to generate a sustained concentration gradient between vasculature and a solid tumor, resulting in significant anticancer effect. To evaluate the impact of molecular weight of the conjugates on treatment efficacy, diblock-, tetrablock- and hexablock GEM and PTX conjugates were administered intravenously to nude mice bearing A2780 human ovarian xenografts. For GEM conjugates, triple doses with dosage 5 mg/kg were given on Days 0, 7, and 14 (q7dx3), whereas single dose regime with 20 mg/kg was applied on Day 0 for PTX conjugates treatment. The most effective conjugates for each monotherapy were the diblock ones, 2P-GEM and 2P-PTX (Mw ~ 100 kDa). Increasing the Mw to 200 or 300 kDa resulted in decrease of activity most probably due to changes in the conformation of the macromolecule because of interaction of hydrophobic residues at side chain termini and formation of "unimer micelles". In addition to monotherapy, a sequential combination treatment of diblock PTX conjugate followed by GEM conjugate (2P-PTX/2P-GEM) were also performed, which showed the best tumor growth inhibition due to synergistic effect-complete remission has been achieved after the first treatment circle. But due to low dose applied, tumor recurrence was observed two weeks after cease of treatment. To assess optimal route of administration, intraperitoneal (i.p.) application of 2P-GEM, 2P-PTX and their combination were examined. The fact that the highest anticancer efficiency was achieved with diblock conjugates that can be synthesized in one scalable step bodes well for the translation into clinics.
- Effect of an enhanced nose-to-brain delivery of insulin on mild and progressive memory loss in the senescence-accelerated mouse. [Journal Article]
- MPMol Pharm 2017 Jan 17
- Insulin is now considered to be a new drug candidate for treating dementias, such as Alzheimer's disease, whose pathologies are linked to insulin resistance in the brain. Our recent work has clarifie...
Insulin is now considered to be a new drug candidate for treating dementias, such as Alzheimer's disease, whose pathologies are linked to insulin resistance in the brain. Our recent work has clarified that a noncovalent strategy involving cell-penetrating peptides (CPPs) can increase the direct transport of insulin from the nasal cavity into the brain parenchyma. The present study aimed to determine whether the brain insulin level increased by intranasal coadministration of insulin with the CPP penetratin has potential for treating dementia. The pharmacological actions of insulin were investigated at different stages of memory impairment using a senescence-accelerated mouse-prone 8 (SAMP8) model. The results of spatial learning tests suggested that chronic intranasal administration of insulin with L-penetratin to SAMP8 slowed the progression of memory loss in the early stage of memory impairment. However, contrary to expectations, this strategy using penetratin was ineffective in recovering the severe cognitive dysfunction in the progressive stage, which involves brain accumulation of amyloid β (Aβ). Immunohistological examination of hippocampal regions of samples from SAMP8 in the progressive stage suggested that accelerated nose-to-brain insulin delivery had a partial neuroprotective function but unexpectedly increased Aβ plaque deposition in the hippocampus. These findings suggest that the efficient nose-to-brain delivery of insulin combined with noncovalent CPP strategy has different effects on dementia during the mild and progressive stages of cognitive dysfunction.
- Preclinical Comparison of Albumin-Binding Radiofolates: Impact of Linker Entities on the in Vitro and in Vivo Properties. [Journal Article]
- MPMol Pharm 2017 Jan 17
- Tumor targeting with folic acid radioconjugates has been proposed as a promising strategy for radionuclide therapy of folate receptor α (FR)-positive cancer. Recently, it was shown that modification ...
Tumor targeting with folic acid radioconjugates has been proposed as a promising strategy for radionuclide therapy of folate receptor α (FR)-positive cancer. Recently, it was shown that modification of radiofolates with an albumin-binding entity increased the tumor-to-kidney ratios of accumulated radioactivity in mice. The goal of this study was to evaluate the lead compound cm10 and compare it with new albumin-binding folate conjugates. Compound cm12 was designed with a long spacer consisting of a PEG-11 entity, and compound cm13 contained a short alkane chain between the albumin-binding moiety and folic acid. All of the derivatives were labeled with (177)Lu (t1/2 = 6.65 days, Eβ(-),average = 134 keV; Eγ = 113 keV, 208 keV), a clinically established radionuclide for therapeutic purposes. The evaluation revealed that all of the albumin-binding radiofolates exhibited increased in vitro stability compared with the reference compound ((177)Lu-cm14) without albumin binder. Serum protein binding, determined with an ultrafiltration assay, was high (>88%) for the derivatives with albumin-binding entities. The FR-binding affinity was in the same range (KD = 4.0-7.5 nM) for all of the radiofolates, independent of the albumin-binding entity and spacer length. FR-specific uptake was proven in vitro using FR-positive KB tumor cells. In vivo studies with KB-tumor-bearing mice were performed in order to assess the tissue distribution profile of the novel radiofolates. (177)Lu-cm13 showed high tumor uptake at late time points (13.3 ± 2.94% IA/g, 48 h p.i.) and tumor-to-kidney ratios (0.59 ± 0.03, 48 h p.i.) in the same range as (177)Lu-cm10 (0.55 ± 0.07, 48 h p.i.). However, the tumor-to-kidney ratio of (177)Lu-cm12 (0.28 ± 0.07, 48 h p.i.) was reduced compared with (177)Lu-cm10 and (177)Lu-cm13. The results of this study indicate that the spacer entity between folic acid and the albumin binder is of critical importance with regard to the tissue distribution profile of the radiofolate. The PEG spacer compromised the beneficial effects of the lead compound, but the design with a short alkane spacer appeared to be promising. Future studies will focus on the design of radiofolates with lipophilic and more rigid spacer entities, which may allow a further improvement of their tissue distribution profiles.
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- Self-assembling peptides epitopes as a novel platform for anti-cancer vaccination. [Journal Article]
- MPMol Pharm 2017 Jan 14
- The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide antig...
The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide antigens in nanoparticulate forms induce stronger immune response than their soluble forms. However, several issues such as poor loading and risk of inducing T cell anergy due to premature release of antigenic epitopes have challenged the clinical success of such systems. In the present study, we developed two vaccine delivery systems by appending a self-assembling peptide (Ac-AAVVLLLW-COOH) or a thermosensitive polymer poly(N-isopropylacrylamide (pNIPAm) to the N-terminus of different peptide antigens (OVA250-264, HPV-E743-57) to generate self-assembling peptide epitopes (SAPEs). The obtained results showed that the SAPEs were able to form nanostructures with an diameter from 20 to 200 nm. The SAPEs adjuvanted with CpG induced and expanded antigen-specific CD8+ T cells in mice. Furthermore, tumor-bearing mice vaccinated with SAPEs harboring the HPV E743-57 peptide showed a delayed tumor growth and an increased survival compared to sham-treated mice. In conclusion, self-assembling peptide based systems increase the immunogenicity of peptide epitope vaccines and therefore warrants further development towards clinical use.