Gary Gellerman

Targeted drug delivery for cancer therapy: the other side of antibodies

Therapeutic monoclonal antibody (TMA) based therapies for cancer have advanced significantly over the past two decades both in their molecular sophistication and clinical efficacy. Initial development efforts focused mainly on humanizing the antibody protein to overcome problems of immunogenicity and on expanding of the target antigen repertoire. In parallel to naked TMAs, antibody-drug conjugates (ADCs) have been developed for targeted delivery of potent anti-cancer drugs with the aim of bypassing the morbidity common to conventional chemotherapy. This paper first presents a review of TMAs and ADCs approved for clinical use by the FDA and those in development, focusing on hematological malignancies. Despite advances in these areas, both TMAs and ADCs still carry limitations and we highlight the more important ones including cancer cell specificity, conjugation chemistry, tumor penetration, product heterogeneity and manufacturing issues. In view of the recognized importance of targeted drug delivery strategies for cancer therapy, we discuss the advantages of alternative drug carriers and where these should be applied, focusing on peptide-drug conjugates (PDCs), particularly those discovered through combinatorial peptide libraries. By defining the advantages and disadvantages of naked TMAs, ADCs and PDCs it should be possible to develop a more rational approach to the application of targeted drug delivery strategies in different situations and ultimately, to a broader basket of more effective therapies for cancer patients.

Recent Innovations in Peptide Based Targeted Drug Delivery to Cancer Cells

Targeted delivery of chemotherapeutics and diagnostic agents conjugated to carrier ligands has made significant progress in recent years, both in regards to the structural design of the conjugates and their biological effectiveness. The goal of targeting specific cell surface receptors through structural compatibility has encouraged the use of peptides as highly specific carriers as short peptides are usually non-antigenic, are structurally simple and synthetically diverse. Recent years have seen many developments in the field of peptide based drug conjugates (PDCs), particularly for cancer therapy, as their use aims to bypass off-target side-effects, reducing the morbidity common to conventional chemotherapy. However, no PDCs have as yet obtained regulatory approval. In this review, we describe the evolution of the peptide-based strategy for targeted delivery of chemotherapeutics and discuss recent innovations in the arena that should lead in the near future to their clinical application.

Synthesis, biological studies and molecular dynamics of new anticancer RGD-based peptide conjugates for targeted drug delivery.

New cyclic RGD peptide-anticancer agent conjugates, with different chemical functionalities attached to the parent peptide were synthesized in order to evaluate their biological activities and to provide a comparative study of their drug release profiles. The Integrin binding c(RGDfK) penta-peptide was used for the synthesis of Camptothecin (CPT) carbamate and Chlorambucil (CLB) amide conjugates. Substitution of the amino acid Lys with Ser resulted in a modified c(RGDfS) with a new attachment site, which enabled the synthesis of an ester CLB conjugate. Functional versatility of the conjugates was reflected in the variability of their drug release profiles, while the conserved RGD sequence of a selective binding to the αv integrin family, likely preserved their recognition by the Integrin and consequently their favorable toxicity towards targeted cancer cells. This hypothesis was supported by a computational analysis suggesting that all conjugates occupy conformational spaces similar to that of the Integrin bound bio-active parent peptide.

Drug resistance to chlorambucil in murine B-cell leukemic cells is overcome by its conjugation to a targeting peptide

Targeting drugs through small-molecule carriers with a high affinity to receptors on cancer cells can overcome the lack of target cell specificity of most anticancer drugs. These targeted carrier–drug conjugates are also capable of reversing drug resistance in cancer cells. Although many targeted drug delivery approaches are being tested, the linkage of several and different drugs to a single carrier molecule might further enhance their therapeutic efficacy, particularly if the drugs are engineered for variable time release. This report shows that murine B-cell leukemic cells previously resistant to a chemotherapeutic drug can be made sensitive to that drug as long as it is conjugated to a targeting peptide and, in particular, when the conjugate contains multiple copies of the drug. Using a 13mer peptide (VHFFKNIVTPRTP) derived from the myelin basic protein (p-MBP), dendrimer-based peptide conjugates containing one, two, or four molecules of chlorambucil were synthesized. Although murine hybridomas expressing antibodies to either p-MBP (MBP cells) or a nonrelevant antigen (BCL-1 cells) were both resistant to free chlorambucil, exposure of the cells to the p-MBP–chlorambucil conjugate completely reversed the drug resistance in MBP, but not BCL-1 cells or normal spleen cells. Moreover, at equivalent drug doses, there was significant enhancement in the cytotoxic activity of multidrug versus single-drug copy conjugates. On the basis of these results, the use of multifunctional dendrone linkers bearing several covalently bound cytotoxic agents allows the development of more effective targeted drug systems and enhances the efficacy of currently approved drugs for B-cell leukemia.

Novel Gly building units for backbone cyclization: synthesis and incorporation into model peptides.

We report the preparation of novel building units for backbone cyclization that have the general formula Fmoc-Nalpha[CH(R)CO2Al]Gly-OH. These building units were prepared by the reductive alkylation method using allyl esters of several amino acids as starting material and hence, respectively, contain the side chain of these amino acids. These N-alkylated Gly building units were incorporated in model backbone cyclic peptides. The resulting crude backbone cyclic peptides were obtained in high degree of purity according to HPLC and mass spectrometric analyses.

Dual‐drug RGD conjugates provide enhanced cytotoxicity to melanoma and non‐small lung cancer cells

To enhance the efficacy of targeted drug delivery, four new peptide‐ligand conjugates were synthesized, each consisting of a cyclic RGDfK penta‐peptide loaded with two anticancer drugs. The drug release profiles in different media of these new compounds and their cytotoxic activity against melanoma and non‐small lung cancer cell lines were evaluated and compared with those of their singly loaded analogs. The cyclic RGDfK penta‐peptide was selected as a targeting moiety because of its high affinity and selectivity to the αvβ3integrin receptor, which is frequently over‐expressed in various types of cancer cells. The peptides core was modified at the side chain of its Lys residue by coupling it with a sixth amino acid (AA) ‐ either Lys (5a) or Ser (5b) (Lys/Ser splitter), resulting in two functional sites which enabled the loading of two therapeutic equivalents onto a single targeting carrier. Using Lys as a splitter resulted in two primary amines. Consequently, conjugates 1a and 1b were synthesized by coupling of 2 Chlorambucils (CLBs) or 2 Camptothecins (CPTs), respectively, to the primary amines of 5a. Conjugate 1c was synthesized from 5b by loading two equivalents of CLB on the amine and the hydroxyl of the Ser splitter, resulting in a homodimeric system with two distinct conjugation sites – amide and ester. The heterodimeric conjugate 1d of CLB and CPT was synthesized by loading each one of the primary amines of 5a with two different drugs ‐ CLB and CPT. The doubling of drug equivalents loaded onto the targeting peptide correlated with enhanced cytotoxic efficacy of the conjugates towards cancer cells. The versatility of chemical linkages of the drugs to the peptides resulted in conjugates with different drug release profiles. Molecular dynamics simulations performed on conjugate 1d demonstrated that this compound occupies a conformational space similar to the bio‐active conformation of an integrin‐bound cyclic RGD peptide reference peptide (c(RGDf(NMe)V). The modified position in 1d (relative to the reference peptide) points away from the integrin, leading us to hypothesize that this peptide binds the integrin in a manner similar to that of the reference peptide thereby fulfilling a crucial requirement for targeted delivery. The strategy of dual drug loading on a single peptide carrier, gives rise to drugs with different mechanisms of action and release profiles, thus substantially increasing the efficacy of selective killing of tumor cells and while reducing the risk of the development of drug resistance.

Facile Synthesis of Orthogonally Protected Optically Pure Keto- and Diketopiperazine Building Blocks for Combinatorial Chemistry

A simple and convenient synthesis of orthogonally protected multi-tethered, optically pure 2-ketopiperazine and 2,5-diketopiperazine scaffolds for Fmoc and Boc combinatorial chemistry was achieved, starting from accessible chiral amino acid precursors, by sequentially utilizing reductive alkylation, dipeptide coupling and ketopiperazine ring formation as key steps. These scaffolds can introduce valuable drug-like properties in three independent directions to any medicinally relevant piperazine-based motif by “around the scaffold” drug optimization. In addition, these building blocks have a wide application scope in managing fast and efficient multi-cyclic optimization processes in the combinatorial chemistry and drug design fields.

Toward the development of a novel non-RGD cyclic peptide drug conjugate for treatment of human metastatic melanoma

The newly discovered short (9 amino acid) non-RGD S-S bridged cyclic peptide ALOS-4 (H-cycl(Cys-Ser-Ser-Ala-Gly-Ser-Leu-Phe-Cys)-OH), which binds to integrin αvβ3 is investigated as peptide carrier for targeted drug delivery against human metastatic melanoma. ALOS4 binds specifically the αvβ3 overexpressing human metastatic melanoma WM-266-4 cell line both in vitro and in ex vivo assays. Coupling ALOS4 to the topoisomerase I inhibitor Camptothecin (ALOS4-CPT) increases the cytotoxicity of CPT against human metastatic melanoma cells while reduces dramatically the cytotoxicity against non-cancerous cells as measured by the levels of γH2A.X, active caspase 3 and cell viability. Moreover, conjugating ALOS4 to CPT even increases the chemo-stability of CPT under physiological pH. Bioinformatic analysis using Rosetta platform revealed potential docking sites of ALOS4 on the αvβ3 integrin which are distinct from the RGD binding sites. We propose to use this specific non-RGD cyclic peptide as the therapeutic carrier for conjugation of drugs in order to improve efficacy and reduce toxicity of currently available treatments of human malignant melanoma.

Anti-proliferative activity of the combination of salan Ti( iv ) complexes with other organic and inorganic anticancer drugs against HT-29 and NCI-H1229 cells: synergism with cisplatin

Two diaminobis(phenolato) Ti(IV) complexes were separately combined with azatoxin, camptothecin or cisplatin and underwent in vitro cytotoxicity analysis toward HT-29 and NCI-H1229 cancer cell lines. The methylated salan derivative exhibited synergistic effects with cisplatin at varying ratios toward both lines, when the compounds were administered fresh and simultaneously, implying great medicinal potential.

Photosensitizer–Antibiotic Conjugates: A Novel Class of Antibacterial Molecules

Bacterial resistance to a variety of antibiotics has led to intensive research into the effect of photosensitizers as a cytotoxic agent against bacterial cells. In this study, we synthesized the following conjugates with or without a linker: rose bengal‐penicillanic acid (RBPA), rose bengal‐linker‐penicillanic acid (RBLPA) and rose bengal‐linker‐kanamycin (RBLKAN). The antibacterial activity of these conjugates was examined on Staphylococcus aureus and Escherichia coli. Exposure of the cultures to 100 J cm−2 showed that the minimum inhibitory concentration (MIC) of RBPA, RBLPA and RBLKAN on S. aureus was 0.195, 0.156 and 0.004 μm, respectively. The MIC of RBPA, RBLPA and RBLKAN on E. coli was 1.56, 2.5 and 0.156 μm, respectively. In dark control experiments, the MIC of these conjugates was not detected until a concentration that was 16‐fold that of the MIC found in the light experiments. RBPA and RBLPA as well as RBLKAN are bactericidal for both bacterial cells. Total eradication of S. aureus and E. coli was observed with RBLKAN (0.078 and 20 μm 16 J cm−2, respectively). Under these conditions, scanning electron microscopic analysis showed significant damage to these bacteria. However, the photosensitizer and antibiotics individually were not effective.