Polylysine Dendrimers Research Articles

Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker.

PEGylation typically improves the systemic exposure and tumor biodistribution of polymeric drug delivery systems, but may also restrict enzyme access to peptide-based drug linkers. The impact of dendrimer generation (G4 vs G5) and PEG length (570 vs 1100 Da) on the pharmacokinetics, tumor biodistribution, drug release kinetics, and anticancer activity of a series of PEGylated polylysine dendrimers conjugated with doxorubicin via a cathepsin-B cleavable valine-citrulline linker was therefore investigated in rodents. Although the smallest G4 PEG570 dendrimer showed the most efficient cathepsin-mediated doxorubicin release, systemic exposure and tumor uptake were limited. The largest G5 PEG1100 dendrimer showed good tumor uptake and retention but restricted drug liberation and therefore limited anticancer activity. Superior anticancer activity was achieved using an intermediate sized dendrimer that showed better drug release kinetics, systemic exposure, tumor uptake, and retention. The data suggest that balancing PEG molecular weight and dendrimer size is critical when designing chemotherapeutic dendrimers.

Doxorubicin Conjugation and Drug Linker Chemistry Alter the Intravenous and Pulmonary Pharmacokinetics of a PEGylated Generation 4 Polylysine Dendrimer in Rats

PEGylated polylysine dendrimers have demonstrated potential as inhalable drug delivery systems that can improve the treatment of lung cancers. Their treatment potential may be enhanced by developing constructs that display prolonged lung retention, together with good systemic absorption, the capacity to passively target lung tumors from the blood and highly selective, yet rapid liberation in the tumor microenvironment. This study sought to characterize how the nature of cathepsin B–cleavable peptide linkers, used to conjugate doxorubicin (Dox) to a PEGylated (PEG570) G4 polylysine dendrimer, affects drug liberation kinetics and intravenous and pulmonary pharmacokinetics in rats. The construct bearing a self-emolative diglycolic acid-V-Citrulline linker exhibited faster Dox release kinetics compared to constructs bearing self-emolative diglycolic acid-glycine-leucine-phenylalanine-glycine (GLFG), or non-self-emolative glutaric acid-GLFG linkers. The V-Citrulline construct exhibited slower plasma clearance, but faster absorption from the lungs than a GLFG construct, although mucociliary clearance and urinary elimination were unchanged. Dox-conjugation enhanced localization in the bronchoalveolar lavage fluid compared to lung tissue, suggesting that projection of Dox from the dendrimer surface reduced tissue uptake. These data show that the linker chemistry employed to conjugate drugs to PEGylated carriers can affect drug release profiles and systemic and lung disposition.

Construction of polylysine dendrimer nanocomposites carrying nattokinase and their application in thrombolysis.

Thrombotic disease has become one of the leading causes of mortality among humans globally. Nattokinase (NK), a novel thrombolytic agent, has attracted the attention of researchers. However, NK is a serine protease that is vulnerable to environmental effects resulting in its inactivation. In this study, polylysine dendrimer (PLLD) was synthesized through divergence-convergence method, and a series of NK/PLLD nanocomposites with different molar ratio was prepared. In addition, NK was successfully incorporated into the cores of PLLD G4 through hydrogen bonds and van der Waals forces. In NK/PLLD nanocomposites, when the molar ratio of NK to PLLD is 1:30, a high relative enzyme activity level (up to 117%) was achieved and was more stable at different temperatures and pH than free NK. In in vitro thrombolysis experiment, compared with free NK, NK/PLLD nanocomposites could control the release of NK. The thrombolysis rate of NK/PLLD nanocomposites reached 50% at 12 h, which can effectively avoid other complications such as hemorrhage. Interestingly, NK/PLLD nanocomposites with positive charge can penetrate into the negatively charged thrombus through electrostatic interaction, thus providing a good thrombolytic effect. Hemolysis and MTT experiments show that PLLD nanomaterials can serve as ideal carriers of protein drugs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 440-449, 2018.

Effect of increased surface hydrophobicity via drug conjugation on the clearance of inhaled PEGylated polylysine dendrimers.

PEGylated polylysine dendrimers are attractive and well tolerated inhalable drug delivery platforms that have the potential to control the release, absorption kinetics and lung retention time of conjugated drugs. The clinical application of these systems though, would likely require partial substitution of surface PEG groups with drug molecules that are anticipated to alter their lung clearance kinetics and clearance pathways. In the current study, we therefore evaluated the impact of increased surface hydrophobicity via substitution of 50% surface PEG groups with a model hydrophobic drug (α-carboxyl OtButylated methotrexate) on the lung clearance of a Generation 5 PEGylated polylysine dendrimer in rats. PEG substitution with OtBu-methotrexate accelerated lung clearance of the dendrimer by increasing polylysine scaffold catabolism, improving systemic absorption of the intact dendrimer and low molecular weight products of scaffold catabolism, and enhancing mucociliary clearance. These results suggest that the conjugation of hydrophobic drug on the surface of a PEGylated dendrimer is likely to accelerate lung clearance when compared to a fully PEGylated dendrimer.

Methotrexate-conjugated PEGylated dendrimers show differential patterns of deposition and activity in tumor-burdened lymph nodes after intravenous and subcutaneous administration in rats.

The current study sought to explore whether the subcutaneous administration of lymph targeted dendrimers, conjugated with a model chemotherapeutic (methotrexate, MTX), was able to enhance anticancer activity against lymph node metastases. The lymphatic pharmacokinetics and antitumor activity of PEGylated polylysine dendrimers conjugated to MTX [D-MTX(OH)] via a tumor-labile hexapeptide linker was examined in rats and compared to a similar system where MTX was α-carboxyl O-tert-butylated [D-MTX(OtBu)]. The latter has previously been shown to exhibit longer plasma circulation times. D-MTX(OtBu) was well absorbed from the subcutaneous injection site via the lymph, and 3 to 4%/g of the dose was retained by sentinel lymph nodes. In contrast, D-MTX(OH) showed limited absorption from the subcutaneous injection site, but absorption was almost exclusively via the lymph. The retention of D-MTX(OH) by sentinel lymph nodes was also significantly elevated (approximately 30% dose/g). MTX alone was not absorbed into the lymph. All dendrimers displayed lower lymph node targeting after intravenous administration. Despite significant differences in the lymph node retention of D-MTX(OH) and D-MTX(OtBu) after subcutaneous and intravenous administration, the growth of lymph node metastases was similarly inhibited. In contrast, the administration of MTX alone did not significantly reduce lymph node tumor growth. Subcutaneous administration of drug-conjugated dendrimers therefore provides an opportunity to improve drug deposition in downstream tumor-burdened lymph nodes. In this case, however, increased lymph node biodistribution did not correlate well with antitumor activity, possibly suggesting constrained drug release at the site of action.

Peptide 612–627 of thyrotropin receptor and its modified analogs as regulators of adenylyl cyclase in rat thyroid gland

The specific activity of the thyroid gland is regulated by thyroid-stimulating hormone (TSH) via TSH receptor (TSHR). This receptor is coupled with various types of G proteins, including Gs proteins, through which TSH stimulates activity of enzyme adenylyl cyclase (AC). Since the use of TSH in medicine is restricted, selective regulators of TSHR with activity of agonists and antagonists are being developed. One approach to their creation is development of peptides corresponding to the functionally important TSHR regions that are located in its cytoplasmic loops and are involved in binding and activation of G proteins. We synthesized peptide corresponding to the C-terminal region 612–627 of the third cytoplasmic loop of TSHR and its derivatives modified by residues of palmitic acid (from the N- or C-termini) or by polylysine dendrimer (from the N-terminus) and we studied their effect on the basal and TSH-stimulated AC activity in membrane fractions isolated from the rat thyroid gland. The most active was peptide 612–627-K(Pal)A modified by palmitate from the C-terminus, where the hydrophobic transmembrane region is located in TSHR. At the micromolar concentrations, it increased AC activity and reduced the AC-stimulating TSH effect. The action of 612–627-K(Pal)A was directed to its homologous TSHR, which is indicated by the following facts: the ingibition of Gs proteins, the transductory component of the AC system, by treatment of the membranes with cholera toxin blocked the AC effect of peptide and this effect was not revealed in the tissues where TSHR are absent, the peptide did not influence the stimulating AC effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer had a much lower ability to activate AC in the thyroid gland than 612–627-K(Pal)A, whereas the peptide modified by palmitate from the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable with 612–627-K(Pal)A by the ability to inhibit the AC action of TSH, but it also decreased (although to the lesser degree) the AC effects of other hormones, which indicates its low receptor specificity. Thereby, the obtained data indicate the high efficiency of the peptide 612–627-K(Pal)A as a regulator of TSHR and the possibilities of creation of drugs on its basis for regulation of thyroid-gland functions in the case of pathology.

Pulmonary administration of a doxorubicin-conjugated dendrimer enhances drug exposure to lung metastases and improves cancer therapy

Direct administration of chemotherapeutic drugs to the lungs significantly enhances drug exposure to lung resident cancers and may improve chemotherapy when compared to intravenous administration. Direct inhalation of uncomplexed or unencapsulated cytotoxic drugs, however, leads to bolus release and unacceptable lung toxicity. Here, we explored the utility of a 56kDa PEGylated polylysine dendrimer, conjugated to doxorubicin, to promote the controlled and prolonged exposure of lung-resident cancers to cytotoxic drug. After intratracheal instillation to rats, approximately 60% of the dendrimer was rapidly removed from the lungs (within 24h) via mucociliary clearance and absorption into the blood. This was followed by a slower clearance phase that reflected both absorption from the lungs (bioavailability 10–13%) and biodegradation of the dendrimer scaffold. After 7days, approximately 15% of the dose remained in the lungs. A syngeneic rat model of lung metastasised breast cancer was subsequently employed to compare the anticancer activity of the dendrimer with a doxorubicin solution formulation after intravenous and pulmonary administration. Twice weekly intratracheal instillation of the dendrimer led to a >95% reduction in lung tumour burden after 2weeks in comparison to IV administration of doxorubicin solution which reduced lung tumour burden by only 30–50%. Intratracheal instillation of an equivalent dose of doxorubicin solution led to extensive lung-related toxicity and death withinseveral days of a single dose. The data suggest that PEGylated dendrimers have potential as inhalable drug delivery systems to promote the prolonged exposure of lung-resident cancers to chemotherapeutic drugs and to improve anti-cancer activity.

PEGylated polylysine dendrimers increase lymphatic exposure to doxorubicin when compared to PEGylated liposomal and solution formulations of doxorubicin

Improved delivery of chemotherapeutic drugs to the lymphatic system has the potential to augment outcomes for cancer therapy by enhancing activity against lymph node metastases. Uptake of small molecule chemotherapeutics into the lymphatic system, however, is limited. Nano-sized drug carriers have the potential to promote access to the lymphatics, but to this point, this has not been examined in detail. The current study therefore evaluated the lymphatic exposure of doxorubicin after subcutaneous and intravenous administration as a simple solution formulation or when formulated as a doxorubicin loaded PEGylated poly-lysine dendrimer (hydrodynamic diameter 12nm), a PEGylated liposome (100nm) and various pluronic micellar formulations (~5nm) to thoracic lymph duct cannulated rats. Plasma and lymph pharmacokinetics were analysed by compartmental pharmacokinetic modelling in S-ADAPT, and Berkeley Madonna software was used to predict the lymphatic exposure of doxorubicin over an extended period of time. The micelle formulations displayed poor in vivo stability, resulting in doxorubicin profiles that were similar to that observed after administration of the doxorubicin solution formulation. In contrast, the dendrimer formulation significantly increased the recovery of doxorubicin in the thoracic lymph after both intravenous and subcutaneous dosing when compared to the solution or micellar formulation. Dendrimer-doxorubicin also resulted in increases in lymphatic doxorubicin concentrations when compared to the liposome formulation, although liposomal doxorubicin did increase lymphatic transport when compared to the solution formulation. Specifically, the dendrimer formulation increased the recovery of doxorubicin in the lymph up to 30h post dose by up to 685 fold and 3.7 fold when compared to the solution and liposomal formulations respectively. Using the compartmental model to predict lymphatic exposure to longer time periods suggested that doxorubicin exposure to the lymphatic system would ultimately be 9796 times and 6.1 times greater after administration of dendrimer doxorubicin when compared to the solution and liposome formulations respectively. The recovery of doxorubicin in the sentinel lymph nodes draining the subcutaneous injection site was also quantified directly, and consistent with the lymph pharmacokinetic data, lymph node recovery was greatest for the dendrimer formulation (12% of dosed doxorubicin/g node) when compared to the liposome (1.4%/g node) and solution (<1%/g node) formulations. The data suggest that dendrimer-based drug delivery systems have the potential to enhance drug exposure to lymph-based drug targets such as lymphatic metastases.

Pulmonary Administration of PEGylated Polylysine Dendrimers: Absorption from the Lung versus Retention within the Lung Is Highly Size-Dependent

The systemic delivery of drugs via the inhaled route is an attractive, needle-free means of improving the systemic exposure of molecules such as peptides and proteins that are poorly absorbed after oral administration. Directed delivery into the lungs also provides a means of increasing drug concentrations at the site of action for lung-specific disease states such as pulmonary infections and lung cancer. The current study has examined the potential utility of PEGylated polylysine dendrimers as pulmonary delivery agents and in particular sought to explore the relationship between dendrimer size and absorption of the intact construct (as a potential systemic delivery mechanism) versus retention within the lungs (as a potential pulmonary depot for controlled local release). Dendrimer absorption from the lungs was inversely correlated with molecular weight, with approximately 20-30% of the dose of relatively small (<22 kDa) dendrimers systemically absorbed compared to only 2% absorption for a larger (78 kDa) PEGylated dendrimer. Increasing the molecular weight of the dendrimers led to slower absorption and more prolonged retention in the lung tissue and bronchoalveolar lavage fluid. Oral administration of the two smaller dendrimers confirmed that oral bioavailability of the PEGylated dendrimers was essentially zero and did not contribute to exposure after pulmonary administration. The smaller PEGylated dendrimers were also degraded in the lungs to low molecular weight products that were subsequently absorbed and excreted via the urine, while the larger constructs showed good stability in the lungs. The data suggest first, that small PEGylated dendrimer-based drug delivery systems may be delivered to the blood via inhalation, providing a more attractive alternative to injections, and second that larger PEGylated dendrimers may be retained in the lungs providing the potential for controlled delivery of medications to the blood or lung tissue.

Synthesis and Evaluation of a Targeted Nanoglobular Dual-Modal Imaging Agent for MR Imaging and Image-Guided Surgery of Prostate Cancer

To synthesize and evaluate a peptide targeted nanoglobular dual modal imaging agent specific to a cancer biomarker in tumor stroma for MRI and fluorescence visualization of prostate tumor in image-guided surgery. A peptide (CGLIIQKNEC, CLT1) targeted generation 2 nanoglobular (polylysine dendrimer with a silsesquioxane core) dual modal imaging agent, CLT1-G2-(Gd-DOTA-MA)-Cy5, was synthesized by stepwise conjugation of Gd-DOTA-MA, Cy5 and peptide to the dendrimer. Contrast enhanced MR imaging of the targeted dual imaging agent was evaluated on a Bruker 7T animal scanner with male athymic nude mice bearing orthotopic PC3-GFP prostate tumor. Fluorescence tumor imaging of the agent was carried out on a Maestro fluorescence imaging system. The targeted agent CLT1-G2-(Gd-DOTA-MA)-Cy5 produced greater contrast enhancement in the tumor tissue than the control agent KAREC-G2-(Gd-DOTA-MA)-Cy5 at a dose of 30 μmol-Gd/kg in the MR images of the tumor bearing mice. Signal-to-noise ratio (SNR) of CLT1-G2-(Gd-DOTA-MA)-Cy5 in the tumor tissue was approximately 2 fold of that of the control agent in the first 15 min post-injection. The targeted agent also resulted in bright fluorescence signals in the tumor tissue. The CLT1 peptide targeted nanoglobular dual-imaging agent CLT1-G2-(Gd-DOTA-MA)-Cy5 has a potential for MRI and fluorescence visualization of prostate tumor.

Molecular Properties of Lysine Dendrimers and their Interactions with A&amp;beta;-Peptides and Neuronal Cells

Prevention of amyloidosis by chemical compounds is a potential therapeutic strategy in Alzheimer’s, prion and other neurodegenerative diseases. Regularly branched dendrimers and less regular hyperbranched polymers have been suggested as promising inhibitors of amyloid aggregation. As demonstrated in our previous studies, some widely used dendrimers (PAMAM, PPI) could not only inhibit amyloid aggregation in solution but also dissolve mature fibrils. In this study we have performed computer simulation of polylysine dendrimers of 3rd and 5th generations (D3 and D5) and analysed the effect of these dendrimers and some hyperbranched polymers on a lysine base (HpbK) on aggregation of amyloid peptide in solution. The effects of dendrimers on cell viability and their protective action against Aβ-induced cytotoxicity and alteration of K+channels was also analysed using human neuroblastoma SH-SY5Y cells. In addition, using fluorescence microscopy, we analysed uptake of FITC-conjugated D3 by SH-SY5Y cells and its distribution in the brain after intraventricular injections to rats. Our results demonstrated that dendrimers D3 and D5 inhibited amyloid aggregation in solution while HpbK enhanced amyloid aggregation. Cell viability and patch-clamp studies have shown that D3 can protect cells against Aβ-induced cytotoxicity and K+channel modulation. In contrast, HpbK had no protective effect against Aβ. Fluorescence microscopy studies demonstrated that FITC-D3 accumulates in the vacuolar compartments of the cells and can be detected in various brain structures and populations of cells after injections to the brain. As such, polylysine dendrimers D3 and D5 can be proposed as compounds for developing antiamyloidogenic drugs. Keywords: Amyloidogenesis, amyloid (Aβ) peptide, cell viability, dendrimers, polylysine dendrimers, patch-clamp, computer simulation, toxicity, fluorescence microscopy, vacuolar compartments

Molecular Properties of Lysine Dendrimers and their Interactions with A&amp;beta;-Peptides and Neuronal Cells

Prevention of amyloidosis by chemical compounds is a potential therapeutic strategy in Alzheimer’s, prion and other neurodegenerative diseases. Regularly branched dendrimers and less regular hyperbranched polymers have been suggested as promising inhibitors of amyloid aggregation. As demonstrated in our previous studies, some widely used dendrimers (PAMAM, PPI) could not only inhibit amyloid aggregation in solution but also dissolve mature fibrils. In this study we have performed computer simulation of polylysine dendrimers of 3rd and 5th generations (D3 and D5) and analysed the effect of these dendrimers and some hyperbranched polymers on a lysine base (HpbK) on aggregation of amyloid peptide in solution. The effects of dendrimers on cell viability and their protective action against Aβ-induced cytotoxicity and alteration of K+channels was also analysed using human neuroblastoma SH-SY5Y cells. In addition, using fluorescence microscopy, we analysed uptake of FITC-conjugated D3 by SH-SY5Y cells and its distribution in the brain after intraventricular injections to rats. Our results demonstrated that dendrimers D3 and D5 inhibited amyloid aggregation in solution while HpbK enhanced amyloid aggregation. Cell viability and patch-clamp studies have shown that D3 can protect cells against Aβ-induced cytotoxicity and K+channel modulation. In contrast, HpbK had no protective effect against Aβ. Fluorescence microscopy studies demonstrated that FITC-D3 accumulates in the vacuolar compartments of the cells and can be detected in various brain structures and populations of cells after injections to the brain. As such, polylysine dendrimers D3 and D5 can be proposed as compounds for developing antiamyloidogenic drugs. Keywords: Amyloidogenesis, amyloid (Aβ) peptide, cell viability, dendrimers, polylysine dendrimers, patch-clamp, computer simulation, toxicity, fluorescence microscopy, vacuolar compartments

Molecular Properties of Lysine Dendrimers and their Interactions with A&amp;beta;-Peptides and Neuronal Cells

Prevention of amyloidosis by chemical compounds is a potential therapeutic strategy in Alzheimer's, prion and other neurodegenerative diseases. Regularly branched dendrimers and less regular hyperbranched polymers have been suggested as promising inhibitors of amyloid aggregation. As demonstrated in our previous studies, some widely used dendrimers (PAMAM, PPI) could not only inhibit amyloid aggregation in solution but also dissolve mature fibrils. In this study we have performed computer simulation of polylysine dendrimers of 3rd and 5th generations (D3 and D5) and analysed the effect of these dendrimers and some hyperbranched polymers on a lysine base (HpbK) on aggregation of amyloid peptide in solution. The effects of dendrimers on cell viability and their protective action against Aβ-induced cytotoxicity and alteration of K+channels was also analysed using human neuroblastoma SH-SY5Y cells. In addition, using fluorescence microscopy, we analysed uptake of FITC-conjugated D3 by SH-SY5Y cells and its distribution in the brain after intraventricular injections to rats. Our results demonstrated that dendrimers D3 and D5 inhibited amyloid aggregation in solution while HpbK enhanced amyloid aggregation. Cell viability and patch-clamp studies have shown that D3 can protect cells against Aβ-induced cytotoxicity and K+channel modulation. In contrast, HpbK had no protective effect against Aβ. Fluorescence microscopy studies demonstrated that FITC-D3 accumulates in the vacuolar compartments of the cells and can be detected in various brain structures and populations of cells after injections to the brain. As such, polylysine dendrimers D3 and D5 can be proposed as compounds for developing antiamyloidogenic drugs.

Abstract 3890: The PhScN peptide as a highly potent inhibitor of basement membrane invasion in vitro and lung colonization in nude mice by metastatic human prostate cancer cells

Abstract Surgery and radiation can cure localized prostate cancer, but not metastatic disease. Our research has shown that activated Δ5α1 integrin fibronectin receptors mediate metastatic invasion by human prostate cancer cell lines, DU 145 and PC-3, as well as angiogenic invasion by human microvascular endothelial cells (hmvec). Thus, activated Δ5α1 receptors are key to metastatic progression. To inhibit metastatic invasion, we devised the Ac-PHSCN-NH2 peptide, PHSCN, licensed as ATN-161. Systemic ATN-161 monotherapy was well tolerated in Phase I trial, and halted metastatic progression for 4-14 months in 35% of patients; however, it was not potent enough for easy administration. Thus, there are some major difficulties with ATN-161 as a drug. Also, PHSCN can be degraded by endoproteinases, enzymes that cleave between its linked L-amino acids. Since tumors secrete abundant endoproteinases, we devised a potent, exo- and endoproteinase-resistant, Δ5α1αtargeted invasion inhibitor, the Ac-PhScN-NH2 peptide (PhScN). Because it contains mirror image D-isomers of His (h) and Cys (c) separating L-isomers of Pro, Ser, and Asn, is resistant to endoproteolytic degradation. Our results suggest that PhScN is 27,000 to 340,000-fold more potent than PHSCN at blocking Δ5α1-mediated, serum-induced, or serum-free PHSRN peptide-induced invasion in vitro by metastatic human DU 145 and PC-3 prostate cancer cells. Our results also indicate that a single pretreatment of suspended DU 145 and PC-3 cells with varying concentrations of the PhScN peptide prior to intravenous injection reduces lung colony formation in athymic, nude mice by 100,000 to 300,000-fold, relative to lung metastasis inhibition by the parental PHSCN peptide. Because Δ5α1 integrin also supports survival, we determined the effects of elevated PhScN concentrations on clonogenic DU 145 and PC-3 survival in vitro. We found that PhScN has no appreciable effect on clonogenic survival, suggesting that its potent anti-metastatic activity is due to invasion inhibition in lung microvasculature. Consistent with this hypothesis, our preliminary results suggest that PhScN pretreatment is also significantly more potent method for preventing extravasation into lung tissue than pretreatment with the parental PHSCN peptide. Since our published results show that the PHSCN polylysine dendrimer (PHSCNGGK-MAP) is 100-fold more potent than the PHSCN peptide at inhibiting DU 145 and PC-3 lung colonization, the results presented here suggest that D-amino acid substitution in the PHSCN sequence, thereby forming the PhScN peptide or dendrimer (PhScNGGK-MAP) may be an excellent strategy for the synthesis of highly potent Δ5α1-targeted inhibitors of metastatic invasion and lung colonization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3890. doi:1538-7445.AM2012-3890

Doxorubicin-Conjugated PEGylated Dendrimers Show Similar Tumoricidal Activity but Lower Systemic Toxicity When Compared to PEGylated Liposome and Solution Formulations in Mouse and Rat Tumor Models

PEGylated polylysine dendrimers show promise as novel drug delivery systems with the potential to direct site specific deposition patterns and to reduce toxicity at nontarget sites. Here the activity and toxicity profiles of a generation 5 polylysine dendrimer with 50% surface conjugation of PEG1100 and 50% surface conjugation of doxorubicin (via an acid labile 4-hydrazinosulfonyl benzoic acid linker) have been compared in a Walker 256 rat tumor model and a human MDA-MB231 xenograft in mice. A direct comparison was also made to a PEGylated liposomal formulation of doxorubicin and a doxorubicin solution. In both rat and mouse breast cancer models, the dendrimer formulation gave equivalent antitumor efficacy when compared to the liposomal or solution doxorubicin formulations and administration of all three doxorubicin formulations resulted in a significant reduction (>75%) in tumor growth in both models at doses ranging from 2 to 10 mg/kg doxorubicin equivalents. The dendrimer formulation, however, was better tolerated by both rats and mice, and approximately 2-fold higher doses were required to induce similar levels of toxicity (as assessed by organ weight, peripheral white cell counts, body weight and survival curves) when compared to administration of the doxorubicin solution or PEGylated liposomal doxorubicin. In rats the appearance of palmar plantar erythematosis (PPE), or hand foot syndrome, was also less evident after administration of dendrimer doxorubicin when compared to the liposome. Finally, even after administration to mice at 2-fold higher doses, dendrimer-doxorubicin resulted in a reduced incidence of cardiotoxicity when compared with a simple solution formulation of doxorubicin. The data suggest that dendrimer-based doxorubicin formulations may provide advantage over solution and liposomal formulations of doxorubicin via a reduction in systemic toxicity.

Macromolecular sensing at the liquid-liquid interface

We report here the electrochemical sensing of macromolecules, such as polyLysine dendrimers, at the polarised liquid | liquid interface. Electrochemistry at the liquid | liquid interface is a powerful analytical technique which allows the detection of non-redox active molecules via ion transfer reactions at a polarised water – oil interface. We demonstrate here that different parameters of the polyLysine dendrimers (charge number, molecular weight) have a strong influence on the sensitivity and limit of detection of these macromolecules. This work will help to the development of sensors based on charge transfer at the liquid | liquid interface.

A comparison of changes to doxorubicin pharmacokinetics, antitumor activity, and toxicity mediated by PEGylated dendrimer and PEGylated liposome drug delivery systems

The pharmacokinetics, biodistribution, and antitumor efficacy of three doxorubicin formulations (doxorubicin in saline, conjugated to a polylysine dendrimer, and encapsulated within a stealth liposome) were investigated in Walker 256 tumor-bearing rats. Liposomal and dendrimer-based delivery systems resulted in more prolonged plasma exposure of total doxorubicin when compared to administration of doxorubicin in saline, although concentrations of free doxorubicin remained low in both cases. Biodistribution profiles revealed enhanced accumulation of dendrimer- and liposome-associated doxorubicin in tumors when compared to doxorubicin alone, although all three doxorubicin formulations reduced tumor growth to a similar extent. Markers of systemic toxicity (spleen weight, white blood cell counts, body weight, and cardiotoxicity) were more pronounced in rats that received doxorubicin and liposomal doxorubicin when compared to dendrimer-doxorubicin. The data provide preliminary evidence that dendrimer-doxorubicin displays similar antitumor efficacy to PEGylated liposomal doxorubicin, but with lower systemic toxicity (resulting from reduced drug exposure to nontarget organs). From the Clinical Editor In this manuscript, three different doxorubicin preparations are compared and preliminary evidence suggests that dendrimer-doxorubicin displays similar antitumor efficacy to PEGylated liposomal doxorubicin, but with lower systemic toxicity.

Characterisation and tumour targeting of PEGylated polylysine dendrimers bearing doxorubicin via a pH labile linker

Polylysine dendrimers have potential as biodegradable vectors for the delivery of cytotoxic drugs to solid tumours. Here, the cytotoxicity, drug release and tumour targeting properties of Generation 5 PEGylated polylysine dendrimers comprising an outer generation of l-lysine or succinimyldipropyldiamine (SPN) and containing doxorubicin (DOX) linked through an acid labile 4-(hydrazinosulfonyl) benzoic acid (HSBA) linker have been characterised. Less than 10% of the DOX load was released from LYS or SPN dendrimers in pH 7.4 buffer over 3 days. In contrast approximately 100% release was evident at pH 5. The DOX-conjugated dendrimers also retained similar cytotoxic properties to free DOX in in vitro cell culture studies (presumably as a result of in situ liberation of free DOX). The clearance patterns of the DOX conjugated SPN and all-lysine dendrimers were similar to the equivalent non-DOX conjugated systems, however the SPN dendrimers showed reduced metabolic lability and increased uptake into RES organs when compared to the equivalent all-lysine dendrimers. In vivo assessment of the DOX-conjugated, PEGylated polylysine dendrimers (both SPN and LYS constructs) in rats bearing Walker 256 tumours revealed higher uptake into tumour tissue when compared with control tissue such as muscle (~ 8 fold) and heart (~ 3 fold). The data suggest that polylysine dendrimers containing DOX conjugated via an acid labile HSBA linker may provide a mechanism to target the delivery of DOX to tumours.

Dendrimer D5 is a Vector for Peptide Transport to Brain Cells

Dendrimers are a new class of nonviral vectors for gene or drug transport. Dendrimer capacity to penetrate through the blood-brain barrier remaines little studied. Biotinylated polylysine dendrimer D5, similarly to human growth hormone biotinylated fragment covalently bound to D5 dendrimer, penetrates through the blood-brain barrier and accumulates in Drosophila brain after injection into the abdomen. Hence, D5 dendrimer can serve as a vector for peptide transport to brain cells.

Capping Methotrexate α-Carboxyl Groups Enhances Systemic Exposure and Retains the Cytotoxicity of Drug Conjugated PEGylated Polylysine Dendrimers

A generation 5 PEGylated (PEG 1100) polylysine dendrimer, conjugated via a stable amide linker to OtBu protected methotrexate (MTX), was previously shown to have a circulatory half-life of 2 days and to target solid tumors in both rats and mice. Here, we show that deprotection of MTX and substitution of the stable linker with a matrix metalloproteinase (MMP) 2 and 9 cleavable linker (PVGLIG) dramatically increased plasma clearance and promoted deposition in the liver and spleen (50-80% of the dose recovered in the liver 3 days post dose). Similar rapid clearance was also seen using a scrambled peptide suggesting that clearance was not dependent on the cleavable nature of the linker. Surprisingly, dendrimers where OtBu capped MTX was linked to the dendrimer surface via the hexapeptide linker showed equivalent in vitro cytotoxicity against HT1080 cells when compared to the uncapped dendrimer and also retained the long circulating characteristics of the stable constructs. The OtBu capped MTX conjugated dendrimer was subsequently shown to significantly reduce tumor growth in HT1080 tumor bearing mice compared to control. In contrast the equivalent dendrimer comprising uncapped MTX conjugated to the dendrimer via the same hexapeptide linker did not reduce tumor growth, presumably reflecting very rapid clearance of the construct. The results are consistent with the suggestion that protection of the α-carboxyl group of methotrexate may be used to improve the circulatory half-life and reduce the liver accumulation of similar MTX-conjugated dendrimers, while still retaining antitumor activity in vivo.