Category: Amino acid/peptide conjugates

The conjugation of different amino acids/peptides to various biologically active compounds has fetched the outstanding results as are very promising drug candidates.

Tumor penetrating peptides for improved drug delivery

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In vivo screening of phage libraries in tumor-bearing mice has been used to identify peptides that direct phage homing to a tumor. The power of in vivo phage screening is illustrated by the recent discovery of peptides with unique tumor-penetrating properties. These peptides activate an endocytic transport pathway related to but distinct from macropinocytosis. They do so through a complex process that involves binding to a primary, tumor-specific receptor, followed by a proteolytic cleavage, and binding to a second receptor. The second receptor, neuropilin-1 (or neuropilin-2) activates the transport pathway. This trans-tissue pathway, dubbed the C-end Rule (CendR) pathway, mediates the extravasation transport through extravascular tumor tissue of payloads ranging from small molecule drugs to nanoparticles. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. Targeted delivery with tumor-penetrating peptides has been shown to specifically increase the accumulation of drugs, antibodies and nanotherapeutics in experimental tumors in vivo, and in human tumors ex vivo. Remarkably the payload does not have to be coupled to the peptide; the peptide activates a bulk transport system that sweeps along a drug present in the blood. Treatment studies in mice have shown improved anti-tumor efficacy and less damage to normal tissues with drugs ranging from traditional chemotherapeutics to antibodies, and to nanoparticle drugs.

The iRGD peptide homes to tumors and accumulates in them through a 3-step process (Fig. 1): First, the integrin-binding RGD sequence motif binds to αvβ3 and αvβ5 integrins, which are specifically expressed in tumor endothelial cells. Other cells in tumors also express these integrins, which is likely to be important for the spreading of the peptide within tumor tissue, but the vascular endothelium is the gateway to the tumor for the peptide. Second, a protease cleavage event activates the CendR motif (R/KXXR/K). This protease(s) has not been identified, but is likely a furin or furin-like enzyme because the CendR motif is a preferred recognition motif for these proteases. In principle, any protease that cuts after a basic residue can activate iRGD. We have used trypsin and urokinase in vitro for this purpose. The protease cleavage requires the integrin binding; a peptide that has the CendR motif but does not bind to integrins (CRG EKGPDC) is not activated. The requirement for integrin binding limits the activation of iRGD to tumors. Third, the CendR motif binds to neuropilin-1 (NRP-1) or neuropilin-2 (NRP-2), and the interaction activates an endocytotic/exocytotic transport pathway named the CendR pathway. This pathway is responsible for the enhanced transport of drugs into tumors triggered by iRGD.

Using an in vivo screening procedure designed to probe tumor lymphatic vessels, we identified a peptide that specifically accumulated in tumor lymphatics and not in normal lymphatics. We now know that this peptide, LyP-1, primarily accumulates in a myeloid cell/macrophage in tumors, when intravenously injected into tumor-bearing mice. Some of these cells incorporate into tumor lymphatics, causing LyP-1 accumulation in the endothelium of these vessels. Endothelial cells of tumor blood vessels and tumor cells also bind LyP-1, but much less of the peptide accumulates in these cells than in tumor macrophages. The macrophages are particularly abundant in hypoxic areas of tumors, which are low on blood vessels but contain abundant, albeit dysfunctional lymphatic vasculature. Remarkably, the phage carrying the LyP-1 peptide reaches these areas within minutes of systemic injection. The ability of this peptide to reach poorly vascularized parts of tumors remained a mystery for several years, until we discovered another peptide with similar tumor-penetrating properties, and set out to uncover the underlying mechanism. (Adv Drug Deliv Rev. 2017 Feb; 110-111: 3–12.)

Targeting the Brain Lesions Using Peptides: A Review Focused on the Possibility of Targeted Drug Delivery to Multiple Sclerosis Lesions

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As described by Jean Martin Charcot in 1868, multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) which leads to permanent disability in patients. Following CNS insults, astrocytes and microglial cells undergo changes, which lead to scar formation in the site of injury. Owning to the pathophysiology of MS lesions, changes in both cellular and extracellular matrix (ECM) components occur over the progression of disease. In spite of advances in therapeutic approaches, drug delivery to MS lesions appears of great interest with big challenges and limitations. Targeting with peptides is a novel promising approach in the field of drug delivery. Recently peptides have been used for active targeting of different pathological disorders in which specific peptides make targeted accumulation of cargos to enhance local drug concentration at the pathological area, lead to increased therapeutic efficacy and decreased side effects. However, specific approaches for targeting the lesion in MS are still lacking. In this review, we discuss the changes of the ECM components as well as the cellular characteristics of demyelinated lesions and emphasis on opportunities for peptide based targeted drug delivery to highlight the possibility of such approaches for neurodegenerative disease with specific focus on MS.

Phthalocyanine–Peptide Conjugates: Receptor-Targeting Bifunctional Agents for Imaging and Photodynamic Therapy

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The synthesis of a series of new zinc phthalocyanine–peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine–glycine–aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine–bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor. (J. Med. Chem. 2013, 56, 4, 1520–1534.)

Synthesis and evaluation of novel dolastatin 10 derivatives for versatile conjugations

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Dolastatin 10 ( buy Pregabalin 75 mg 1) is a highly potent cytotoxic microtubule inhibitor (cytotoxicity IC50 < 5.0 nM) and several of its analogs have recently been used as payloads in antibody drug conjugates. Herein, we describe the design and synthesis of a series of novel dolastatin 10 analogs useful as payloads for conjugated drugs. We explored analogs containing functional groups at the thiazole moiety at the C-terminal of dolastatin 10. The functional groups included amines, alcohols, and thiols, which are representative structures used in known conjugated drugs. These novel analogs showed excellent potency in a tumor cell proliferation assay, and thus this series of dolastatin 10 analogs is suitable as versatile payloads in conjugated drugs. Insights into the structure–activity relationships of the analogs are also discussed.(Bioorganic & Medicinal Chemistry. Volume 26, Issue 8, 1 May 2018, Pages 1643-1652.)

Esters of terpene alcohols as highly potent, reversible, and low toxic skin penetration enhancers

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Skin penetration/permeation enhancers are compounds that improve (trans)dermal drug delivery. We designed hybrid terpene-amino acid enhancers by conjugating natural terpenes (citronellol, geraniol, nerol, farnesol, linalool, perillyl alcohol, menthol, borneol, carveol) or cinnamyl alcohol with 6-(dimethylamino)hexanoic acid through a biodegradable ester linker. The compounds were screened for their ability to increase the delivery of theophylline and hydrocortisone through and into human skin ex vivo. The citronellyl, bornyl and cinnamyl esters showed exceptional permeation-enhancing properties (enhancement ratios up to 82) while having low cellular toxicities. The barrier function of enhancer-treated skin (assessed by transepidermal water loss and electrical impedance) recovered within 24 h. Infrared spectroscopy suggested that these esters fluidized the stratum corneum lipids. Furthermore, the citronellyl ester increased the epidermal concentration of topically applied cidofovir, which is a potent antiviral and anticancer drug, by 15-fold. In conclusion, citronellyl 6-(dimethylamino)hexanoate is an outstanding enhancer with an advantageous combination of properties, which may improve the delivery of drugs that have a limited ability to cross biological barriers.

Terpenes are a class of natural compounds with strong permeation-enhancing potential and have been generally recognized as safe (GRAS) adjuvants with relatively low and transient irritation. For example, the acyclic monoterpene alcohols citronellol, geraniol, and linalool, enhanced the permeation of ondansetron, caffeine and haloperidol, respectively. The cyclic monoterpenes borneol, carveol, menthol, and limonene were reported as enhancers for ibuprofen, curcumin, indomethacin, and valsartan, respectively. In addition, sesquiterpene farnesol increased the permeation of haloperidol. (Sci Rep. 2019; 9: 14617.)

N-terminus FITC labeling of peptides on solid support: the truth behind the spacer

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Fluorescein isothiocyanate (FITC) is an amine reactive derivative of fluorescein dye that has wide ranging application in biochemistry. It has been extensively used to label peptides and proteins. However, its use in solid phase peptide synthesis is restricted. Indeed, in acidic conditions required for linker cleavage, N-ter FITC-labeled peptides undergo a cyclization leading to the formation of a fluorescein with subsequent removal of the last amino acid. This can be avoided when a spacer such as amino hexanoic acid is used or if non-acidic cleavage is operated to release targeted peptide from the resin. (Tetrahedron Letters. Volume 50, Issue 3, 21 January 2009, Pages 260-263.)

Effects of Chemical Conjugation of l-Leucine to Chitosan on Dispersibility and Controlled Release of Drug from a Nanoparticulate Dry Powder Inhaler Formulation

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This study investigated l-leucine-conjugated chitosan as a drug delivery vehicle in terms of dispersibility and controlled release from a nanoparticulate dry powder inhaler (DPI) formulation for pulmonary delivery using diltiazem hydrochloride (DH) as the model drug. DH-loaded nanoparticles of chitosan and conjugate were prepared by water-in-oil emulsification followed by glutaraldehyde cross-linking. Nanoparticles were characterized by dynamic light scattering for particle size, X-ray photoelectron spectroscopy for surface composition, and twin stage impinger for drug dispersibility. The controlled release of DH was studied in phosphate-buffered saline (pH 7.3 ± 0.2, 37 °C) using UV spectrophotometry. The fine particle fractions of conjugated chitosan with and without drug were higher than those of nonconjugated chitosan nanoparticles. The conjugate nanoparticles were superior to those of unmodified chitosan in drug loading, entrapment efficiency, and controlled release profile. The higher dispersibility was attributed to the amphiphilic environment of the l-leucine conjugate and hydrophobic cross-links, and the release profile reflects the greater swelling. The conjugated chitosan nanoparticles could be useful, after appropriate testing for biodegradability and toxicity, as an alternative carrier for lung drug delivery with enhanced aerosolization and prolonged drug release from nanoparticulate DPI formulations. (Mol Pharm. 2016 May 2;13(5):1455-66. )