Micelles based on HPMA copolymers

Polymeric micelles have been under extensive investigation during the past years as drug delivery systems, particularly for anticancer drugs. They are formed by the self-assembly of amphiphilic block copolymers in aqueous solutions and have a spherical shape and a size in the nano-range (< 200 nm). Tumor accumulation of polymeric micelles upon intravenous administration can occur as a result of the leaky vasculature of tumor tissue (called the enhanced permeation and retention (EPR) effect).To benefit from the EPR effect, polymeric micelles need to have prolonged circulation times as well as high and stable drug loadings. Poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA) is a hydrophilic polymer currently under investigation for its use in polymer-drug conjugates. Its biocompatibility, non-immunogenicity and the possibility for functionalization are properties that resulted in broad pharmaceutical and biomedical applications, also in the micelle technology research. Being hydrophilic, it can serve as a micellar stealth corona, while it can also be modified with hydrophobic moieties to serve as a micellar core in which hydrophobic drugs can be solubilized and retained. HPMA-based polymeric micelles have been showing very promising in vitro and in vivo results. This review summarizes the applications of pHPMA in the field of polymeric micelles, either serving as a micellar stealth corona, or, if hydrophobically rendered by derivatization, as a micellar core.     

HPMA copolymers: Origins, early developments, present, and future

The overview covers the discovery of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, initial studies on their synthesis, evaluation of biological properties, and explorations of their potential as carriers of biologically active compounds in general and anticancer drugs in particular. The focus is on the research in the authors' laboratory — the development of macromolecular therapeutics for the treatment of cancer and musculoskeletal diseases. In addition, the evaluation of HPMA (co)polymers as building blocks of modified and new biomaterials is presented: the utilization of semitelechelic poly(HPMA) and HPMA copolymers for the modification of biomaterial and protein surfaces and the design of hybrid block and graft HPMA copolymers that self-assemble into smart hydrogels. Finally, suggestions for the design of second-generation macromolecular therapeutics are portrayed.     

Reducible HPMA-co-oligolysine copolymers for nucleic acid delivery

Biodegradability can be incorporated into cationic polymers via use of disulfide linkages that are degraded in the reducing environment of the cell cytosol. In this work, N-(2-hydroxypropyl)methacrylamide (HPMA) and methacrylamido-functionalized oligo-l-lysine peptide monomers with either a non-reducible 6-aminohexanoic acid (AHX) linker or a reducible 3-[(2-aminoethyl)dithiol] propionic acid (AEDP) linker were copolymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Both of the copolymers and a 1:1 (w/w) mixture of copolymers with reducible and non-reducible peptides were complexed with DNA to form polyplexes. The polyplexes were tested for salt stability, transfection efficiency, and cytotoxicity. The HPMA-oligolysine copolymer containing the reducible AEDP linkers was less efficient at transfection than the non-reducible polymer and was prone to flocculation in saline and serum-containing conditions, but was also not cytotoxic at charge ratios tested. Optimal transfection efficiency and toxicity were attained with mixed formulation of copolymers. Flow cytometry uptake studies indicated that blocking extracellular thiols did not restore transfection efficiency and that the decreased transfection of the reducible polyplex is therefore not primarily caused by extracellular polymer reduction by free thiols. The decrease in transfection efficiency of the reducible polymers could be partially mitigated by the addition of low concentrations of EDTA to prevent metal-catalyzed oxidation of reduced polymers.     

N-(2-羟丙基)甲基丙烯酰胺聚合物-5-氟尿嘧啶接合物的体外释药规律、体内分布及抗肿瘤活性研究

考察本实验室合成的N-(2-羟丙基)甲基丙烯酰胺［N-(2-hydroxypropyl) methacrylamide，HPMA］聚合物-5-氟尿嘧啶(5-flurouracil，5-FU)接合物(P-FU)的体外释药、体内分布及抗肿瘤活性。以小鼠血浆为介质，考察P-FU中5-FU的释放规律；以小鼠H22肝癌实体瘤模型(皮下型)为肿瘤模型，考察接合物在荷瘤小鼠体内的分布情况、药代动力学规律及抑瘤活性。结果表明，37 ℃时P-FU在小鼠血浆中具有一定的稳定性，半衰期(t_1/2)为32.4 h。与5-FU相比，P-FU在荷瘤小鼠体内的循环时间明显延长(血浆中t_1/2为原药的166倍)，在肿瘤中的沉积量(AUC为5-FU的3.3倍)及滞留时间(t_1/2为5-FU的2.3倍)均有明显增加。体内药效学研究表明，P-FU组对荷瘤小鼠的肿瘤生长抑制率(69.09%)显著高于5-FU组(56.49%，P<0.05)，瘤块组织病理学观察结果也显示P-FU组小鼠肿瘤组织中细胞凋亡程度大于5-FU组。HPMA聚合物可被用于为5-FU构建一种新型实体瘤高分子给药系统。     

In vitro cytotoxicity, in vivo biodistribution and antitumor activity of HPMA copolymer–5-fluorouracil conjugates

5-Fluorouracil (5-FU) is an antimetabolite with a broad-spectrum activity against solid tumors. However, its very short half-life in plasma circulation greatly limited the in vivo antitumor efficacy and clinical application. The current work aimed to solve this problem as well as to increase 5-FU biodistribution to tumor by covalently conjugating 5-FU to a biocompatible, non-toxic and non-immunogenic drug carrier – N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer. The in vitro cytotoxicity, in vivo biodistribution and therapeutic efficacy of HPMA copolymer–5-FU conjugates (P-FU) were reported. Cytotoxicity was evaluated by using a serial of tumor cells (A549, CT-26, Hela, HepG₂ cells and 5-FU resistant HepG₂ cells). In vivo biodistribution and therapeutic efficacy were investigated in Kunming mice-bearing hepatoma 22 (H₂₂). Results indicated that P-FU could increase the cytotoxicity of 5-FU in Hela, HepG₂ and 5-FU resistant HepG₂ cells, while it decreases the cytotoxicity of 5-FU in A549 and CT-26. Both in vitro release profile in plasma and biodistribution study showed that P-FU significantly prolonged the drug plasma circulation time. P-FU also showed an over 3-fold larger area under the concentration–time curve (AUC) in tumor when compared with free drug. Therapeutic evaluation also demonstrated that the treatment with P-FU displayed stronger inhibition of the tumor growth when compared with that of control group (physiologic saline) or 5-FU group at the same dose. All the results suggested that P-FU could increase cytotoxicity of 5-FU in certain cancer cell lines, prolong 5-FU circulation time in vivo, enhance 5-FU distribution to tumor and improve therapeutic efficacy. Therefore, HPMA copolymer is a potential carrier for 5-FU for the effective treatment of cancer.     

Polymeric Drug-Carriers Containing Doxorubicin and Melanocyte-Stimulating Hormone: In Vitro and In Vivo Evaluation Against Murine Melanoma

Abstract

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing doxorubicin (DOX, approximately 8% by weight) bound via the lysosomally degradable spacer Gly-Phe-Leu-Gly and, in certain cases, also melanocyre-stimulating hormone (MSH, 5-10% by weight) were synthesized with the aim of developing a drug conjugate for site-specific delivery to malignant melanoma. Polymer-bound MSH, like free MSH, was able to stimulate tyrosinase activity in B16F10 cells in vitro, confirming the ability of conjugated hormone to interact with the MSH receptor. Similarly, a ¹²⁵I-labelled conjugate containing MSH was captured by B16F10 cells in vitro more rapidly than a similar polymer without the targeting moiety. HPMA copolymers containing DOX bound via the lysosomally degradable Gly-Phe-Leu-Gly linkage were cytotoxic to a mouse melanoma cell line (M3 S91) in vitro, the MSH-containing conjugate being more active than that without (although the difference in the ID₅₀ was not significant). When administered intraperitoneally or intravenously to C57BL/6J mice bearing intraperitoneal B16F10 tumours, HPMA copolymers containing DOX linked via this biodegradable spacer (with or without MSH) significantly increased animal survival, the maximum ratio of the mean survival of the test group (T) to that of the untreated control (C) T/C observed (approximately 200) over the dose range 5-20 mg DOX/kg being similar to that seen for free DOX. In contrast, neither polymer conjugates containing DOX bound via a non-degradable linkage (Gly-Gly) nor free MSH showed antitumour activity. In mice bearing established subcutaneous B16F10 tumours, biodegradable polymer-bound DOX conjugates given intraperitoneally were more effective than free DOX (which was virtually inactive in this system); conjugates containing MSH were significantly more effective than those without, the maximum T/C being approximately 148 and 324 respectively. Preliminary pharmacokinetic experiments showed evidence of selective MSH targeting of polymer conjugates to subcutaneous B16F10.     

N-(2-羟丙基)甲基丙烯酰胺聚合物-5-氟尿嘧啶接合物的体外释药规律、体内分布及抗肿瘤活性研究

考察本实验室合成的N-(2-羟丙基)甲基丙烯酰胺[N-(2-hydroxypropyl)methacrylamide,HPMA]聚合物-5-氟尿嘧啶(5-flurouracil,5-FU)接合物(P-FU)的体外释药、体内分布及抗肿瘤活性。以小鼠血浆为介质,考察P-FU中5-FU的释放规律;以小鼠H22肝癌实体瘤模型(皮下型)为肿瘤模型,考察接合物在荷瘤小鼠体内的分布情况、药代动力学规律及抑瘤活性。结果表明,37℃时P-FU在小鼠血浆中具有一定的稳定性,半衰期(t1/2)为32.4 h。与5-FU相比,P-FU在荷瘤小鼠体内的循环时间明显延长(血浆中t1/2为原药的166倍),在肿瘤中的沉积量(AUC为5-FU的3.3倍)及滞留时间(t1/2为5-FU的2.3倍)均有明显增加。体内药效学研究表明,P-FU组对荷瘤小鼠的肿瘤生长抑制率(69.09%)显著高于5-FU组(56.49%,P<0.05),瘤块组织病理学观察结果也显示P-FU组小鼠肿瘤组织中细胞凋亡程度大于5-FU组。HPMA聚合物可被用于为5-FU构建一种新型实体瘤高分子给药系统。     

Noninvasive Visualization of Pharmacokinetics,Biodistribution and Tumor Targeting of Poly[<Emphasis Type="BoldItalic">N</Emphasis>-(2-hydroxypropyl)methacrylamide] in Mice Using Contrast Enhanced MRI

Purpose To study a non-invasive method of using contrast enhanced magnetic resonance imaging (MRI) to visualize the real-time pharmacokinetics, biodistribution and tumor accumulation of paramagnetically labeled poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) copolymer conjugates with different molecular weights and spacers in tumor-bearing mice. Materials and Methods Paramagnetically labeled HPMA copolymer conjugates were synthesized by free radical copolymerization of HPMA with monomers containing a chelating ligand, followed by complexation with Gd(OAc)₃. A stable paramagnetic chelate, Gd-DO3A, was conjugated to the copolymers via a degradable spacer GlyPheLeuGly and a non-degradable spacer GlyGly, respectively. The conjugates with molecular weights of 28, 60 and 121 kDa and narrow molecular weight distributions were prepared by fractionation with size exclusion chromatography. The conjugates were injected into athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts via a tail vein. MR images were acquired before and at various time points after the injection with a 3D FLASH sequence and a 2D spin-echo sequence at 3T. Pharmacokinetics, biodistribution and tumor accumulation of the conjugates were visualized based on the contrast enhancement in the blood, major organs and tumor tissue at various time points. The size effect of the conjugates was analyzed among the conjugates. Results Contrast enhanced MRI resulted in a real-time, three-dimensional visualization of blood circulation, pharmacokinetics, biodistribution and tumor accumulation of the conjugates, and the size effect on these pharmaceutical properties. HPMA copolymer conjugates with high molecular weight had a prolonged blood circulation time and high passive tumor targeting efficiency. Non-biodegradable HPMA copolymers with molecular weights higher than the threshold of renal filtration demonstrated higher efficiency for tumor drug delivery than biodegradable poly(L-glutamic acid). Conclusions Contrast enhanced MRI is an effective method for non-invasive visualization of in vivo properties of the paramagnetically labeled polymer conjugates in preclinical studies.     

Poly [N -(2-hydroxypropyl)methacrylamide] Conjugates of Bovine Pancreatic Ribonuclease (RNase A) Inhibit Growth of Human Melanoma in Nude Mice

Recently hydrophilic poly [N -(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 μ g/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125 I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24 h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.     

Correlation of Subcellular Compartmentalization of HPMA Copolymer-Mce6 Conjugates with Chemotherapeutic Activity in Human Ovarian Carcinoma Cells

Purpose. Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e₆ monoethylenediamine (Mce₆) conjugates was explored to aid in the design of second-generation PDT delivery systems. Methods. Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce₆ was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce₆, P-NLS(FITC)-GG-Mce₆) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce₆, P-NLS(FITC)-GFLG-Mce₆). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC₅₀), cell associated drug concentration (CAD) and confocal microscopy. Results. P-GFLG-Mce₆ possessed enhanced chemotherapeutic activity compared to P-GG-Mce₆ indicating enzymatically released Mce₆ was more active than copolymer-bound Mce₆. Lysosomes appeared less sensitive to photodamage as observed by a higher IC₅₀. Nuclear-directed HPMA copolymer-Mce₆ conjugates (P-NLS(FITC)-GG-Mce₆ P-NLS(FITC)-GFLG-Mce₆) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce₆ conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce₆) or amino groups (P-NH₂-GG-Mce₆) also displayed increased chemotherapeutic activity. Conclusions. Nuclear delivery was observed for P-NLS(FITC)-GG-Mce₆ and P-NLS(FITC)-GFLG-Mce₆ indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.     

Natural and synthetic poly(malic acid)-based derivates: a family of versatile biopolymers for the design of drug nanocarriers

Abstract

The field of specific drug delivery is an expanding research domain. Besides the use of liposomes formed from various lipids, natural and synthetic polymers have been developed to prepare more efficient drug delivery systems either under macromolecular prodrugs or under particulate nanovectors. To ameliorate the biocompatibility of such nanocarriers, degradable natural or synthetic polymers have attracted the interest of many researchers. In this context, poly(malic acid) (PMLA) extracted from microorganisms or synthesized from malic or aspartic acid was used to prepare water-soluble drug carriers or nanoparticles. Within this review, both the preparation and the applications of PMLA derivatives are described emphasizing the in vitro and in vivo assays. The results obtained by several groups highlight the interest of such polyesters in the field of drug delivery.     

N-homolupinanoyl and N-(omega-lupinylthio)alkanoyl derivatives of some tricyclic systems as ligands for muscarinic M1 and M2 receptor subtypes

A set of N-homolupinanoyl- and N-(omega-lupinylthio)alkanoyl derivatives of tricyclic systems (as phenothiazine, iminodibenzyl and dihydropyridobenzodiazepinone) has been prepared and tested for affinity for rat muscarinic M(1) and M(2) receptor subtypes labeled with [3H]pirenzepine and [3H]AF-DX 384. Good affinity for both M(1) and M(2) subtypes was displayed by most compounds, often with nanomolar K(i) values, which for lupinylthiopropionyl- and lupinylthiobutyryl-phenothiazines (13-16) were comparable to those of pirenzepine and methoctramine, respectively. However, only moderate selectivity for one or the other subtype was seen.     

Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety

We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2'- and 3'-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2'-OH being more essential. Thus, the 2'-fluoro substitution eliminated both binding and activation, while a 3'-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5'-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3'-fluoro derivatives. The 4'-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5'-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1'- to 4'-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a K(B) value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.     

Investigations on the cytochrome P450 (CYP) isoenzymes involved in the metabolism of the designer drugs N-(1-phenyl cyclohexyl)-2-ethoxyethanamine and N-(1-phenylcyclohexyl)-2-methoxyethanamine

Investigations using insect cell microsomes with cDNA-expressed human cytochrome P450 (CYP)s and human liver microsomes (HLM) are reported on the CYP isoenzymes involved in the metabolism of the designer drugs N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA) to O-deethyl PCEEA and N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) to O-demethyl PCMEA. Gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry was used for the analysis of the incubation samples. PCEEA O-deethylation was catalyzed by CYP2B6, CYP2C9, CYP2C19, and CYP3A4, while PCMEA O-demethylation was catalyzed only by CYP2B6 and CYP2C19. Considering the relative activity factor approach, these enzymes accounted for 53%, 25%, 4%, and 18% of net clearance for PCEEA and 91% and 9% of net clearance for PCMEA, respectively. The chemical CYP2B6 inhibitor 4-(4-chlorobenzyl)pyridine (CBP) reduced the metabolite formation in pooled HLM by 63% at 1 μM PCEEA. At 10 μM PCEEA, CBP reduced metabolite formation by 61%, while inhibition of CYP3A4 by ketoconazole and inhibition of CYP2C9 by sulfaphenazole showed no inhibitory effect. At 1 μM PCMEA, CBP reduced metabolite formation in pooled HLM by 70% and at 10 μM PCMEA by 78%, respectively. In conclusion, the main metabolic step of both studied drugs was catalyzed by different CYPs.     

Evaluation and modification of N-trimethyl chitosan chloride nanoparticles as protein carriers

N-Trimethyl chitosan chloride (TMC) nanoparticles were prepared by ionic crosslinking of TMC with tripolyphosphate (TPP). Two model proteins with different pI values, bovine serum albumin (BSA, pI=4.8) and bovine hemoglobin (BHb, pI=6.8), were used to investigate the loading and release features of the TMC nanoparticles. TMC samples with different degrees of quaternization were synthesized to evaluate its influence on the physicochemical properties and release profiles of the nanoparticles. Sodium alginate was used to modify the TMC nanoparticles to reduce burst release. The results indicated that the TMC nanoparticles had a high loading efficiency (95%) for BSA but a low one (30%) for BHb. The particle size and zeta potential were significantly affected by the BSA concentration but not by the BHb concentration. Nanoparticles of TMC with a lower degree of quaternization showed an increase in particle size, a decrease in zeta potential and a slower drug-release profile. As for the alginate-modified nanoparticles, a smaller size and lower zeta potential were observed and the burst release of BSA was reduced. These studies demonstrated that TMC nanoparticles are potential protein carriers, and that their physicochemical properties and release profile could be optimized by means of various modifications.     

Synthesis and antifungal activity of new N-(1-phenyl-4-carbetoxypyrazol-5-yl)-, N-(indazol-3-yl)- and N-(indazol-5-yl)-2-iodobenzamides

N-(1-Phenyl-4-carbetoxypyrazol-5-yl)-, N-(indazol-3-yl)- and N-(indazol-5-yl)-2-iodobenzamides 6, with a Benodanil-like structure, were synthesized by refluxing in acetic acid the corresponding benzotriazinones 5 with potassium iodide for 1 h in order to study the role on the antifungal activity of the N-substitution with an aromatic heterocyclic system on benzamide moiety. Among the tested iododerivatives, compounds 6d,f,g,h possess interesting activities toward some phytopathogenic fungal strains.     

Methylated N-(4-pyridinylmethyl) chitosan as a novel effective safe gene carrier

The objective of this study was to study the transfection efficiency of quaternized N-(4-pyridinylmethyl) chitosan; TM-Py-CS, using the pDNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh 7 cells). The factors affecting the transfection efficiency, e.g. degree of quaternization (DQ), the extent of N-pyridinylmethyl substitution (ES) and weight ratio, have been investigated. The results revealed that TM-Py-CS was able to condense with pDNA. Illustrated by agarose gel electrophoresis, complete complexes of TM(69)Py(62)CS/DNA were formed at weight ratio above 1.1, whereas those of TM(53)Py(40)CS/DNA and TM(52)Py(13)CS/DNA were above 1.8 and 8, respectively. TM(69)Py(62)CS showed superior transfection efficiency to TM(53)Py(40)CS, TM(52)Py(13)CS, TM(65)CS and TM(43)CS at all weight ratios tested. The highest transfection efficiency of TM(69)Py(62)CS/DNA complexes was found at weight ratio of 4. The results indicated that the improved gene transfection was possibly due to 4-pyridinylmethyl substitution on CS which promoted the interaction and condensation with DNA as well as N-quaternization which increased CS water solubility. In cytotoxicity studies, high concentration of TM-Py-CS and TM-CS could decrease the Huh 7 cell viability. In conclusion, this novel CS derivative, TM(69)Py(62)CS, showed promising potential as a gene carrier by efficient DNA condensation and mediated higher level of gene transfection.     

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant’s leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.     

Safety and efficacy of sodium caprate in promoting oral drug absorption: from in vitro to the clinic

A major challenge in oral drug delivery is the development of novel dosage forms to promote absorption of poorly permeable drugs across the intestinal epithelium. To date, no absorption promoter has been approved in a formulation specifically designed for oral delivery of Class III molecules. Promoters that are designated safe for human consumption have been licensed for use in a recently approved buccal insulin spray delivery system and also for many years as part of an ampicillin rectal suppository. Unlike buccal and rectal delivery, oral formulations containing absorption promoters have the additional technical hurdle whereby the promoter and payload must be co-released in high concentrations at the small intestinal epithelium in order to generate significant but rapidly reversible increases in permeability. An advanced promoter in the clinic is the medium chain fatty acid (MCFA), sodium caprate (C₁₀), a compound already approved as a food additive. We discuss how it has evolved to a matrix tablet format suitable for administration to humans under the headings of mechanism of action at the cellular and tissue level as well as in vitro and in vivo efficacy and safety studies. In specific clinical examples, we review how C₁₀-based formulations are being tested for oral delivery of bisphosphonates using Gastro Intestinal Permeation Enhancement Technology, GIPET® (Merrion Pharmaceuticals, Ireland) and in a related solid dose format for antisense oligonucleotides (ISIS Pharmaceuticals, USA).     

HPMA copolymer-cyclic RGD conjugates for tumor targeting

This review describes the design and development of N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-cyclic RGD conjugates for targeting tumor angiogenesis. Relative to non-targetable systems, HPMA copolymer-RGD4C and -RGDfK conjugates have shown increased tumor accumulation in a variety of solid tumors including prostate, lung, and breast tumor xenografts. Compared to free peptides, copolymers had increased tumor accumulation and decreased uptake in non-target organs such as the liver and spleen. Clinically relevant imaging agents such as ^99mTc, ¹¹¹In, and Gd enabled in vivo imaging of the constructs by scintigraphy and magnetic resonance techniques. Targeted delivery of ⁹⁰Y, a radiotherapeutic agent by HPMA copolymer-RGD4C conjugates resulted in tumor size reduction in mice bearing prostate tumor xenografts. Delivery of the geldanamycin derivative 17-(6-aminohexylamino)-17-demethoxygeldanamycin by HPMA copolymer-RGDfK conjugates resulted in increased tumor concentration of the free drug in a prostate xenograft model. These constructs show promise for targeted delivery of therapeutics and imaging agents to solid tumors.