Anti-cancer peptides (ACPs) are small cationic and hydrophobic peptides that are more toxic to cancer cells than normal cells. ACPs kill cancer cells by causing irreparable membrane damage and cell ...lysis, or by inducing apoptosis. Direct-acting ACPs do not bind to a unique receptor, but are rather attracted to several different molecules on the surface of cancer cells. Here we report that an amidated wasp venom peptide, Mastoparan, exhibited potent anti-cancer activities toward leukemia (IC50~8–9.2μM), myeloma (IC50~11μM), and breast cancer cells (IC50~20–24μM), including multidrug resistant and slow growing cancer cells. Importantly, the potency and mechanism of cancer cell killing was related to the amidation of the C-terminal carboxyl group. Mastoparan was less toxic to normal cells than it was to cancer cells (e.g., IC50 to PBMC=48μM). Mastoparan killed cancer cells by a lytic mechanism. Moreover, Mastoparan enhanced etoposide-induced cell death in vitro. Our data also suggest that Mastoparan and gemcitabine work synergistically in a mouse model of mammary carcinoma. Collectively, these data demonstrate that Mastoparan is a broad-spectrum, direct-acting ACP that warrants additional study as a new therapeutic agent for the treatment of various cancers.
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•Mastoparan-NH2 is a broad-spectrum, direct-acting, anti-cancer peptide.•Mastoparan potency and mechanism of action is related to its C-terminal amidation.•Mastoparan enhances etoposide-induced cytotoxicity in vitro.•Systemically-delivered Mastoparan and gemcitabine may work synergistically in vivo.
Zein can be utilized to form nanoscale particles for drug delivery applications. Despite the ease of synthesis, these particles often aggregate when exposed to physiologically relevant conditions ...(e.g., pH and salt concentrations). This instability has prevented their further development in applications requiring intravenous administration. To mitigate this colloidal instability, this research explored Zein nanoparticles (NP)s that were modified with polyethylene glycol (PEG) either through functionalized PEG pre- or post-NP formation. The results suggest that the pre-functionalization of the Zein using N-hydroxysuccinimide ester terminated PEG is the method of choice for synthesizing Zein NPs with conjugated PEG (Zein:PEG-Zein NPs). Zein:PEG-Zein NPs formed using this method displayed excellent stability in physiologically relevant conditions over 72 h and were stable at 4 °C for at least 3 months. When the NPs were cultured with cells for 72 h, no cytotoxicity or early signs of apoptosis were identified. Cellular uptake of the Zein:PEG-Zein NPs did not seem to be impacted by the amount of PEG incorporated in the NP but were concentration-, time-, and temperature-dependent. The lowest percent, stable Zein:PEG-Zein NP formulation (80% unmodified Zein and 20% PEG-modified Zein) induced no observable toxicity over 14 days in CD-1 mice dosed at 70 mg/kg via the tail vein. However, repeat dose pharmacokinetic (PK) studies demonstrated that following the first dose, the second dose caused health issues that required euthanasia shortly after administration. For those animals that survived, there was faster plasma elimination of the Zein:PEG-Zein NPs. Despite this, the Zein:PEG-Zein NPs represent a significantly improved formulation approach, one that displays a long circulation half-life and is suitable for single-use administration. Repeat dose applications will require additional methods to silence the immune response that is generated when using these NPs intravenously.
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of ...SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation
and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.
The Ru(III) complexes indazolium trans-RuCl4(1H-indazole)2 (KP1019) and sodium trans-RuCl4(1H-indazole)2 (NKP-1339) are leading candidates for the next generation of metal-based chemotherapeutics. ...Trifluoromethyl derivatives of these compounds and their imidazole and pyridine analogues were synthesized to probe the effect of ligand lipophilicity on the pharmacological properties of these types of complexes. Addition of CF3 groups also provided a spectroscopic handle for 19F NMR studies of ligand exchange processes and protein interactions. The lipophilicities of the CF3-functionalized compounds and their unsubstituted parent complexes were quantified by the shake-flask method to give the distribution coefficient D at pH 7.4 (log D 7.4). The solution behavior of the CF3-functionalized complexes was characterized in phosphate-buffered saline (PBS) using 19F NMR, electron paramagnetic resonance (EPR), and UV–vis spectroscopies. These techniques, along with fluorescence competition experiments, were also used to characterize interactions with human serum albumin (HSA). From these studies it was determined that increased lipophilicity correlates with reduced solubility in PBS but enhancement of noncoordinate interactions with hydrophobic domains of HSA. These protein interactions improve the solubility of the complexes and inhibit the formation of oligomeric species. EPR measurements also demonstrated the formation of HSA-coordinated species with longer incubation. 19F NMR spectra show that the trifluoromethyl complexes release axial ligands in PBS and in the presence of HSA. In vitro testing showed that the most lipophilic complexes had the greatest cytotoxic activity. Addition of CF3 groups enhances the activity of the indazole complex against A549 nonsmall cell lung carcinoma cells. Furthermore, in the case of the pyridine complexes, the parent compound was inactive against the HT-29 human colon carcinoma cell line but showed strong cytotoxicity with CF3 functionalization. Overall, these studies demonstrate that lipophilicity may be a determining factor in the anticancer activity and pharmacological behavior of these types of Ru(III) complexes.
A passive equilibration method which relies on addition of candidate drugs to pre-formed liposomes is described as an alternative method for preparing liposome encapsulated drugs. The method is ...simple, rapid and applicable to liposomes prepared with high (45mol%) or low (<20mol%) levels of cholesterol. Passive equilibration is performed in 4-steps: (i) formation of liposomes, (ii) addition of the candidate drug to the liposomes in combination with a permeability enhancing agent, (iii) incubation at a temperature that facilitates diffusion of the added compound across the lipid bilayer, and (iv) quenching the enhanced membrane permeability by reduction in temperature and/or removal of the permeabilization enhancer. The method is fully exemplified here using ethanol as the permeabilization enhancer and carboplatin (CBDCA) as the drug candidate. It is demonstrated that ethanol can be added to liposomes prepared with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and Cholesterol (Chol) (55:45mol ratio) in amounts up to 30% (v/v) with no change in liposome size, even when incubated at temperatures>60°C. Super-saturated solutions of CBDCA (40mg/mL) can be prepared at 70°C and these are stable in the presence of ethanol even when the temperature is reduced to <30°C. maximum CBDCA encapsulation is achieved within 1h after the CBDCA solution is added to pre-formed DSPC/Chol liposomes in the presence of 30% (v/v) ethanol at 60°C. When the pre-formed liposomes are mixed with ethanol (30% v/v) at or below 40°C, the encapsulation efficiency is reduced by an order of magnitude. The method was also applied to liposomes prepared from other compositions include a cholesterol free formulations (containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)-2000 (DSPE-PEG2000)) and a low Chol (<20mol%) formulations prepared with the distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) DSPG)). The cytotoxic activity of CBDCA was unaffected when prepared in this manner and two of the resultant formulations exhibited good stability in vitro and in vivo. The cytotoxic activity of CBDCA was unaffected when prepared in this manner and the resultant formulations exhibited good stability in vitro and in vivo. Pharmacokinetics studies in CD-1 mice indicated that the resulting formulations increased the circulation half life of the associated CBDCA significantly (AUC0–24h of CBDCA=0.016μg·hr/mL; AUC0-24h of the DSPC/Chol CBDCA formulation=1014.0μg·hr/mL and AUC0-24h of the DSPC/DSPG/Chol CBDCA formulation=583.96μg·hr/mL). Preliminary efficacy studies in Rag-2M mice with established subcutaneous H1975 and U-251 tumors suggest that the therapeutic activity of CBDCA is improved when administered in liposomal formulations. The encapsulation method described here has not been disclosed previously and will have broad applications to drugs that would normally be encapsulated during liposome manufacturing.
Liposomal loading of carboplatin through passive encapsulation and passive equilibration. Display omitted
For more than 30 years, treatment of acute myeloid leukemia (AML) has remained largely unchanged and reliant on chemotherapeutic drug combinations, specifically cytarabine and daunorubicin (the 7 + 3 ...regimen). One broad spectrum drug, flavopiridol (also known as Alvocidib) has shown significant activity against AML through the inhibition of cyclin-dependent kinases. Flavopiridol is a semisynthetic flavonoid and our research team recently described methods to formulate another flavonoid, quercetin, through the ability of flavonoids to bind divalent metals. This method relies on use of copper-containing liposomes to enhance the apparent solubility of flavopiridol and to create formulations suitable for intravenous (i.v.) use. Similar to quercetin, flavopiridol is defined as an aqueous-insoluble compound (< 1 mg/mL in water) and this research sought to evaluate whether the copper-binding capabilities of flavopiridol could be used to prepare an injectable formulation that would exhibit enhanced exposure and improved efficacy. Flavopiridol powder was added directly to preformed copper-containing liposomes (DSPC:Chol or DSPC:DSPE-PEG2000) and the resulting formulations were characterized. Pharmacokinetic and efficacy studies were then conducted. The liposomal flavopiridol formulations were well-tolerated in mice following i.v. administration at a dose of 5 mg/kg with no apparent acute or chronic toxicities. In vivo pharmacokinetics of the optimized DSPC/DSPE-PEG2000 liposomal flavopiridol formulation demonstrated a 30-fold increase in AUC (0.804 μg-hr/mL versus 26.92 μg-hr/mL) compared to the free flavopiridol formulation. The resultant liposomal formulation also demonstrated significant therapeutic activity in MV4–11 and MOLM-13 subcutaneous AML models. Additional studies will be required to define whether formulation changes can be made to enhance flavopiridol retention in the selected composition. The results suggest that further increases in flavopiridol retention will result in improved therapeutic activity.
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As a tribute to Pieter R. Cullis, this manuscript identifies a liposomal formulation that bears his initials: the PRCosomes. "Pretty" Reactive Complexes within liposomes were observed, while the ...senior author of this manuscript completed his Ph.D. thesis under Pieter's supervision. The dye (safranine) was used as a tool to measure the magnitude of the transmembrane gradient generated with liposomes. The dye's redistribution is easily detected by eye and correlates with >98% encapsulation of the dye. This observation became the basis from which remote drug loading methods developed. Remote loading methodology involves the addition of drugs to pre-formed liposomes with a transmembrane gradient, which results in drug redistribution to the liposome interior. Doxorubicin, as an example drug candidate, complexes manganese trapped within the liposome. A color change accompanied drug encapsulation as the solution went from an orange to purple. This manuscript reviews and adds a novel perspective on the use of metal complexation reactions to prepare PRCosomes. The technology described provides a versatile method to form metal-drug complexed within liposomes. The purpose of this work is to differentiation between drug candidate loading that is caused by metal-drug complexation and loading driven by formation of a pH gradient.
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Therapeutic monoclonal antibodies hold great promise in the treatment of cancer and other diseases, but their unclear mechanism of action makes it difficult to identify features that ...will increase their efficacy. One such feature may be antibody valence, since enhanced therapeutic efficacies have been observed using multivalent, as opposed to bivalent, antibodies. For example, multivalent antibody-lipid nanoparticles (Ab-LNPs) containing rituximab (Rtx) or trastuzumab show significantly increased therapeutic activity compared to equivalent doses of the bivalent antibodies. To more fully understand this phenomenon, we created a methodology reliant on biotin-neutravidin interactions for preparing specific valences of Ab-LNPs that shows improvements in reproducibility, preparation time and overall yield of coupled Ab (up to 80%). We subsequently prepared a series of valences of Rtx-LNPs to examine binding characteristics to CD20+ lymphoma cells, distribution of Rtx-LNPs on the cell surface, modulation of CD20 expression, cytotoxicity of the constructs and ability of the different valences to directly induce apoptosis. As the valence of Rtx-LNP was increased, the amount of Rtx bound to cells increased up to ∼10-fold higher compared to bivalent Rtx. Although more Rtx was bound to cells, there were also surprising increases in the levels of unbound CD20. This suggested the formation of Rtx-enriched microdomains that were confirmed using confocal fluorescence laser-scanning microscopy. Multivalent Rtx-LNPs were significantly more cytotoxic than Rtx; for equivalent doses of drug, Rtx-LNPs elicited apoptosis in two lymphoma cell lines in a valence-dependent manner up to levels that were 14-fold higher than bivalent Rtx. It is suggested that CD20-enriched microdomains may play a role in the mechanism of action of Rtx. This new preparation methodology can be used in future studies evaluating the mechanism of action of multivalent Ab-LNPs prepared with Rtx or other therapeutic Abs.
Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a ...mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.
Immunotherapy has changed the way many cancers are being treated. Researchers in the field of immunotherapy and tumor immunology are investigating similar questions: How can the positive benefits ...achieved with immunotherapies be enhanced? Can this be achieved through combinations with other agents and if so, which ones? In our view, there is an urgent need to improve immunotherapy to make further gains in the overall survival for those patients that should benefit from immunotherapy. While numerous different approaches are being considered, our team believes that drug delivery methods along with appropriately selected small-molecule drugs and drug candidates could help reach the goal of doubling the overall survival rate that is seen in some patients that are given immunotherapeutics. This review article is prepared to address how immunotherapies should be combined with a second treatment using an approach that could realize therapeutic gains 10 years from now. For context, an overview of immunotherapy and cancer angiogenesis is provided. The major targets in angiogenesis that have modulatory effects on the tumor microenvironment and immune cells are highlighted. A combination approach that, for us, has the greatest potential for success involves treatments that will normalize the tumor’s blood vessel structure and alter the immune microenvironment to support the action of immunotherapeutics. So, this is reviewed as well. Our focus is to provide an insight into some strategies that will engender vascular normalization that may be better than previously described approaches. The potential for drug delivery systems to promote tumor blood vessel normalization is considered.
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