Month: March 2021

Tumor penetrating peptides for improved drug delivery

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 (CRGEKGPDC) 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.)

Synthesis and Pharmacological Effects of Diosgenin–Betulinic Acid Conjugates

The target diosgenin–betulinic acid conjugates are reported to investigate their ability to enhance and modify the pharmacological effects of their components. The detailed synthetic procedure that includes copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click reaction), and palladium-catalyzed debenzylation by hydrogenolysis is described together with the results of cytotoxicity screening tests. Palladium-catalyzed debenzylation reaction of benzyl ester intermediates was the key step in this synthetic procedure due to the simultaneous presence of a 1,4-disubstituted 1,2,3-triazole ring in the molecule that was a competing coordination site for the palladium catalyst. High pressure (130 kPa) palladium-catalyzed procedure represented a successful synthetic step yielding the required products. The conjugate 7 showed selective cytotoxicity in human T-lymphoblastic leukemia (CEM) cancer cells (IC50 = 6.5 ± 1.1 µM), in contrast to the conjugate 8 showing no cytotoxicity, and diosgenin (1), an adaptogen, for which a potential to be active on central nervous system was calculated in silico. In addition, 5 showed medium multifarious cytotoxicity in human T-lymphoblastic leukemia (CEM), human cervical cancer (HeLa), and human colon cancer (HCT 116). Betulinic acid (2) and the intermediates 3 and 4 showed no cytotoxicity in the tested cancer cell lines. The experimental data obtained are supplemented by and compared with the in silico calculated physico-chemical and absorption, distribution, metabolism, and excretion (ADME) parameters of these compounds.

Diosgenin, (3β,25R)-spirost-5-en-3-ol, is a steroid sapogenin part of the saponin dioscin found in the tubers of Dioscorea zingiberensis C. H. Wright or Trigonella foenum-graecum L. and in numbers of legumes. Diosgenin is a widely used precursor in the synthesis of sexual hormones, peroral contraceptives and other steroids in the pharmaceutical industry. It is an adaptogen, displaying non-steroidogenic activity along with other beneficial effects. Diosgenin is unable to bind metal ions, and therefore, the change made from more traditional cholesterol/cholesterylamine system to diosgenin could influence the overall conformation of the bivalent structures, modifying the metal ions chelating properties. Saponins are always species formed from an aglycone and several monosaccharide units, the presence of which increases the solubility of saponins in natural aqueous media. Diosgenin is not metabolized in the human body, and it is considered to represent a safe natural drug. It has also been investigated for treating hyperglycemia, hypercholesterolemia, hypertriacylglycerolemia, and Alzheimer’s disease.
Betulinic acid, 3β-hydroxylup-20(29)-en-28-oic acid, is a pharmacologically perspective triterpenoid plant product with a broad spectrum of effects, e.g., antitumor, anti-HIV, cytostatic, and anti-inflammatory. It can be obtained from the bark of Betula pendula Roth, widely distributed in Europe, and from a number of subtropical and tropical plants. (Molecules. 2020 Aug; 25(15): 3546.)

Mitocanic Di- and Triterpenoid Rhodamine B Conjugates

The combination of the “correct” triterpenoid, the “correct” spacer and rhodamine B (RhoB) seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, several triterpenoid rhodamine B conjugates have been prepared and screened for their cytotoxic activity. To obtain cytotoxic compounds with EC50 values in a low nano-molar range combined with good tumor/non-tumor selectivity, the Rho B unit has to be attached via an amine spacer to the terpenoid skeleton. To avoid spirolactamization, secondary amines have to be used. First results indicate that a homopiperazinyl spacer is superior to a piperazinyl spacer. Hybrids derived from maslinic acid or tormentic acid are superior to those from oleanolic, ursolic, glycyrrhetinic or euscaphic acid. Thus, a tormentic acid-derived RhoB conjugate 32, holding a homopiperazinyl spacer can be regarded, at present, as the most promising candidate for further biological studies.

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Mitochondrial membranes of malignant cells hold an increased membrane potential compared to non–malignant cells. This effect fosters the accumulation of cationic molecules, hence inducing high selectivity for mitocans holding a (more or less) lipophilic cation such as a rhodamine scaffold. The same effect applies for triphenylphosphonium cations and to a small extent for quaternary ammonium ions, zwitterionic N-oxides and triterpenes substituted with BODIPYs or a safirinium moiety [67].
To date, hybrid molecules have been prepared from oleanolic acid (OA, Figure 2), ursolic acid (UA), glycyrrhetinic acid (GA), betulinic acid (BA), maslinic acid (MA), augustic acid (AU), 11-keto-β-boswellic acid (KBA), asiatic acid (AA), tormentic acid (TA) and euscaphic acid (EA).
OA-derived RhoB conjugates appear to be superior to analog UA-derived compounds in the majority of cases with respect to their cytotoxicity. Although AKBA-derived derivatives have good cytotoxicity properties, they were found to be less cytotoxic compared to other triterpene carboxylic acid derivatives, but they often showed better tumor cell/non-tumor cell selectivity. So far, the best cytotoxicity properties have been found for MA-, EA- and TA-derived derivatives. These allowed the transition to compounds of nano-molar activity, while many other triterpene carboxylic acid derivatives were cytotoxic only on a micro-molar concentration range. MA- derived derivatives seem to be approximately equivalent to EA-derived compounds. They are currently only surpassed in many tumor cell lines only by the analogous derivatives from TA. From results available so far, it can be concluded that compounds holding a homopiperazinyl spacer are superior to those with a piperazinyl spacer. This underlines the importance of the spacer for obtaining good cytotoxicity properties. (Molecules. 2020 Nov; 25(22): 5443.)