Vincent J. Venditto

Cancer Nanomedicines: So Many Papers and So Few Drugs!

This review identifies a timeline to nanomedicine anticancer drug approval using the business model of inventors, innovators and imitators. By evaluating the publication record of nanomedicine cancer therapeutics we identified a trend of very few publications prior to FDA approval. We first enumerated the publications related to cancer involving polymers, liposomes or monoclonal antibodies and determined the number of citations per publication as well as the number of published clinical trials among the publications. Combining these data with the development of specific nanomedicines, we are able to identify an invention phase consisting of seminal papers in basic science necessary for the development of a specific nanomedicine. The innovation phase includes the first report, the development and the clinical trials involving that nanomedicine. Finally, the imitation phase begins after approval when others ride the wave of success by using the same formulation for new drugs or using the same drug to validate other nanomedicines. We then focused our analysis on nanomedicines containing camptothecin derivatives, which are not yet approved including two polymers considered innovations and one liposomal formulation in the imitation phase. The conclusion that may be drawn from the analysis of the camptothecins is that approved drugs reformulated in polymeric and liposomal cancer nanomedicines have a more difficult time navigating through the approval process than the parent molecule. This is probably due to the fact that for most currently approved drugs, reformulating them in a nanocarrier provides a small increase in performance that large pharmaceutical companies do not consider being worth the time, effort and expense of development. It also appears that drug carriers have a more difficult path through the clinic than monoclonal antibodies. The added complexity of nanocarriers also deters their use to deliver new molecular entities. Thus, the new drug candidates that might be most improved by drug delivery in nanocarriers are not formulated in this fashion.

Cancer therapies utilizing the camptothecins: a review of the in vivo literature.

This review summarizes the in vivo assessment-preliminary, preclinical, and clinical-of chemotherapeutics derived from camptothecin or a derivative. Camptothecin is a naturally occurring, pentacyclic quinoline alkaloid that possesses high cytotoxic activity in a variety of cell lines. Major limitations of the drug, including poor solubility and hydrolysis under physiological conditions, prevent full clinical utilization. Camptothecin remains at equilibrium in an active lactone form and inactive hydrolyzed carboxylate form. The active lactone binds to DNA topoisomerase I cleavage complex, believed to be the single site of activity. Binding inhibits DNA religation, resulting in apoptosis. A series of small molecule camptothecin derivatives have been developed that increase solubility, lactone stability and bioavailability to varying levels of success. A number of macromolecular agents have also been described wherein camptothecin(s) are covalently appended or noncovalently associated with the goal of improving solubility and lactone stability, while taking advantage of the tumor physiology to deliver larger doses of drug to the tumor with lower systemic toxicity. With the increasing interest in drug delivery and polymer therapeutics, additional constructs are anticipated. The goal of this review is to summarize the relevant literature for others interested in the field of camptothecin-based therapeutics, specifically in the context of biodistribution, dosing regimens, and pharmacokinetics with the desire of providing a useful source of comparative data. To this end, only constructs where in vivo data is available are reported. The review includes published reports in English through mid-2009.

Designer lipids for drug delivery: From heads to tails

For four decades, liposomes composed of both naturally occurring and synthetic lipids have been investigated as delivery vehicles for low molecular weight and macromolecular drugs. These studies paved the way for the clinical and commercial success of a number of liposomal drugs, each of which required a tailored formulation; one liposome size does not fit all drugs! Instead, the physicochemical properties of the liposome must be matched to the pharmacology of the drug. An extensive biophysical literature demonstrates that varying lipid composition can influence the size, membrane stability, in vivo interactions, and drug release properties of a liposome. In this review we focus on recently described synthetic lipid headgroups, linkers and hydrophobic domains that can provide control over the intermolecular forces, phase preference, and macroscopic behavior of liposomes. These synthetic lipids further our understanding of lipid biophysics, promote targeted drug delivery and improve liposome stability. We further highlight the immune reactivity of novel synthetic headgroups as a key design consideration. For instance it was originally thought that synthetic PEGylated lipids were immunologically inert; however, its been observed that under certain conditions PEGylated lipids induce humoral immunity. Such immune activation may be a limitation to the use of other engineered lipid headgroups for drug delivery. In addition to the potential immunogenicity of engineered lipids, future investigations on liposome drugs in vivo should pay particular attention to the location and dynamics of payload release.

The 8 year thicket of triazine dendrimers: strategies, targets and applications

This manuscript focuses on the routes, methods and reagents used to synthesize triazine-based dendrimers. Our pursuit of macromolecular architectures for drug delivery—dendrimers based on triazines—has been an ongoing effort for 8 years. To date, we have produced complex dendrimers with diverse peripheries as proof-of-concept, less complex molecules tailored for specific applications including DNA and RNA delivery and drug-decorated dendrimers for potential therapeutic applications including infectious disease and cancer. These syntheses have been executed at scales that range from high milligrams to over a kilogram. The routes, reagents and diversity displayed by a target anchors it in time. Early targets derive from convergent synthetic routes while later targets are prepared using divergent syntheses. The core of early dendrimers was a simple diamine, including piperazine, yielding the so-called bow-tie structures, middle period targets boast either a trispiperazinyltriazine core or a ‘super-core’ with six piperazine groups. Later targets return to the trispiperazinyltriazine core. The choice of linking diamine has also changed. Over time, p-aminobenzylamine was replaced by piperazine and then by aminomethylpiperidine with more exotic diamines sprinkled in throughout. Peripheral group choice has undergone similar variations: from AB2 to AB4 to, more recently, AB3. The diversity communicated by these groups yields dendrimers ranging from those with a common surface to examples where two groups were presented to those where four orthogonally reactive groups appear. Over time, these groups have grown in complexity from protected amines to tags for biodistribution and drugs like paclitaxel. Herein, strategies adopted and lessons learned are reviewed, intuitions relayed and future directions forecast.

The Effect of Polymer Backbone Chemistry on the Induction of the Accelerated Blood Clearance in Polymer Modified Liposomes

A variety of water-soluble polymers, when attached to a liposome, substantially increase liposome circulation half-life in animals. However, in certain conditions, liposomes modified with the most widely used polymer, polyethylene glycol (PEG), induce an IgM response resulting in an accelerated blood clearance (ABC) of the liposome upon the second injection. Modification of liposomes with other water-soluble polymers: HPMA (poly[N-(2-hydroxypropyl) methacrylamide]), PVP (poly(vinylpyrrolidone)), PMOX (poly(2-methyl-2-oxazoline)), PDMA (poly(N,N-dimethyl acrylamide)), and PAcM (poly(N-acryloyl morpholine)), increases circulation times of liposomes; but a precise comparison of their ability to promote long circulation or induce the ABC effect has not been reported. To obtain a more nuanced understanding of the role of polymer structure/MW to promote long circulation, we synthesized a library of polymer diacyl chain lipids with low polydispersity (1.04-1.09), similar polymer molecular weights (2.1-2.5kDa) and incorporated them into 100nm liposomes of a narrow polydispersity (0.25-1.3) composed of polymer-lipid/hydrogenated soy phosphatidylcholine/cholesterol/diD: 5.0/54.5/40/0.5. We confirm that HPMA, PVP, PMOX, PDMA and PAcM modified liposome have increased circulation times in rodents and that PVP, PDMA, and PAcM do not induce the ABC effect. We demonstrate for the first time, that HPMA does not cause an ABC effect whereas PMOX induces a pronounced ABC effect in rats. We find that a single dose of liposomes coated with PEG and PMOX generates an IgM response in rats towards the respective polymer. Finally, in this homologous polymer series, we observe a positive correlation (R=0.84 in rats, R=0.92 in mice) between the circulation time of polymer-modified liposomes and polymer viscosity; PEG and PMOX, the polymers that can initiate an ABC response were the two most viscous polymers. Our findings suggest that polymers that do not cause an ABC effect such as, HPMA or PVP, deserve further consideration as polymer coatings to improve the circulation of liposomes and other nanoparticles.

Antibody Response to Polyhistidine-Tagged Peptide and Protein Antigens Attached to Liposomes via Lipid-Linked Nitrilotriacetic Acid in Mice

ABSTRACT Particulate delivery systems enhance antibody responses to subunit antigens. However, covalent attachment of protein antigens can disrupt protein structure and mask critical epitopes, altering the antibody response to the antigen. In this report, we evaluate noncovalent metal chelation via nitrilotriacetic acid (NTA) as a nondestructive method to attach peptide and protein antigens to liposomes. Two model antigens, ovalbumin (OVA) and a peptide derived from the membrane-proximal region of HIV-1 gp41 (N-MPR), were polyhistidinylated and attached to liposomes via monovalent NTA (mono-NTA; KD [equilibrium dissociation constant], ∼10 μM), trivalent NTA (tris-NTA; KD, ∼1 nM), or a covalent linkage. Attachment of N-MPR, but not OVA, to liposomes via an NTA lipid elicited stronger antibody responses in BALB/c mice than a formulation in which unassociated antigen was simply admixed with control liposomes lacking NTA. However, the tris-NTA linkage did not increase antibody responses to either N-MPR or OVA compared to the level for the mono-NTA linkage, despite the greater liposomal association of the antigen. For both antigens, covalently attaching them to a lipid elicited significantly stronger antibody responses than NTA-anchored antigens (OVA titer, 3.4 × 106 versus 1.4 × 106 to 1.6 × 106 [P < 0.001]; N-MPR titer, 4.4 × 104 versus 5.5 × 102 to 7.6 × 102 [P < 0.003]). The data indicate that NTA linkages may increase antibody titers to weak antigens such as N-MPR, but NTA-mediated attachment remains inferior to covalent conjugation. Moreover, enhancements in antigen-liposome affinity do not result in increased antibody titers. Thus, additional improvements of NTA-mediated conjugation technology are necessary to achieve an effective, nondestructive method for increasing the humoral response to antigens in particulate vaccines.

Unraveling the mechanism of cell death induced by chemical fibrils

We previously discovered a small-molecule inducer of cell death, named 1541, that non-covalently self-assembles into chemical fibrils (“chemi-fibrils”) and activates procaspase-3 in vitro. We report here that 1541-induced cell death is caused by the fibrillar, rather than the soluble form of the drug. An shRNA screen reveals that knockdown of genes involved in endocytosis, vesicle trafficking, and lysosomal acidification causes partial 1541 resistance. We confirm the role of these pathways using pharmacological inhibitors. Microscopy shows that the fluorescent chemi-fibrils accumulate in punctae inside cells that partially co-localize with lysosomes. Notably, the chemi-fibrils bind and induce liposome leakage in vitro, suggesting they may do the same in cells. The chemi-fibrils induce extensive proteolysis including caspase substrates, yet modulatory profiling reveals that chemi-fibrils form a distinct class from existing inducers of cell death. The chemi-fibrils share similarities to proteinaceous fibrils and may provide insight into their mechanism of cellular toxicity.

Intercepting the synthesis of triazine dendrimers with nucleophilic pharmacophores: a general strategy toward drug delivery vehicles.

The camptothecin ester of isonipecotic acid is installed on a triazine dendrimer intermediate obtained through an iterative, scalable route to ultimately yield cationic and PEGylated targets with activities in cell culture comparable to free drug.

Rational Design of Membrane Proximal External Region Lipopeptides Containing Chemical Modifications for HIV-1 Vaccination

ABSTRACT The inability to generate broadly neutralizing antibody (bnAb) responses to the membrane proximal external region (MPER) of HIV-1 gp41 using current vaccine strategies has hampered efforts to prevent the spread of HIV. To address this challenge, we investigated a novel hypothesis to help improve the anti-MPER antibody response. Guided by structural insights and the unique lipid reactivity of anti-MPER bnAbs, we considered whether amino acid side chain modifications that emulate hydrophilic phospholipid head groups could contribute to the generation of 2F5-like or 4E10-like neutralizing anti-MPER antibodies. To test this hypothesis, we generated a series of chemically modified MPER immunogens through derivatization of amino acid side chains with phosphate or nitrate groups. We evaluated the binding affinity of the chemically modified peptides to their cognate monoclonal antibodies, 2F5 and 4E10, using surface plasmon resonance. The modifications had little effect on binding to the antibodies and did not influence epitope secondary structure when presented in liposomes. We selected five of the chemically modified sequences to immunize rabbits and found that an immunogen containing both the 2F5 and 4E10 epitopes and a phosphorylated threonine at T676 elicited the highest anti-peptide IgG titers, although the high antipeptide titers did not confer higher neutralizing activity. These data indicate that side chain modifications adjacent to known neutralizing antibody epitopes are capable of eliciting antibody responses to the MPER but that these chemically modified gp41 epitopes do not induce neutralizing antibodies.

Chemically Modified Peptides Based on the Membrane-Proximal External Region of the HIV-1 Envelope Induce High-Titer, Epitope- Specific Nonneutralizing Antibodies in Rabbits

ABSTRACT Broadly neutralizing monoclonal antibodies (bNAbs) 2F5 and 4E10 bind to the membrane proximal external region (MPER) of gp41 and also cross-react with phospholipids. In this study, we investigated if chemical modifications on the MPER adjacent to 2F5 and 4E10 epitopes using mimetics of inflammation-associated posttranslational modifications to induce 2F5- and 4E10-like bNAbs can break tolerance. We synthesized a series of chemically modified peptides spanning the MPER. The serine, threonine, and tyrosine residues in the peptides were modified with sulfate, phosphate, or nitrate moieties and presented in liposomes for rabbit immunizations. All immunizations resulted in high antisera titers directed toward both the modified and unmodified immunogens. Tyrosine modification was observed to significantly suppress antiepitope responses. Sera with strong anti-gp140 titers were purified by affinity chromatography toward the MPER peptide and found to possess a higher affinity toward the MPER than did the bNAbs 2F5 and 4E10. Modest neutralization was observed in the H9 neutralization assay, but neutralization was not observed in the TZM-bl cell or peripheral blood mononuclear cell (PBMC) neutralization assay platforms. Although neutralizing antibodies were not induced by this approach, we conclude that chemical modifications can increase the immune responses to poorly immunogenic antigens, suggesting that chemical modification in an appropriate immunization protocol should be explored further as an HIV-1 vaccine strategy.