Development of New Gonadotropin-Releasing Hormone-Modified

Article Cite This: J. Med. Chem. 2017, 60, 8309-8320

pubs.acs.org/jmc

Development of New Gonadotropin-Releasing Hormone-Modified Dendrimer Platforms with Direct Antiproliferative and Gonadotropin Releasing Activity Pegah Varamini,†,∥,¶ Amirreza Rafiee,†,∥,# Ashwini Kumar Giddam,† Friederike M. Mansfeld,†,∇ Frederik Steyn,‡ and Istvan Toth*,†,§,⊥ †

School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland 4072, Australia The University of Queensland Centre for Clinical Research and the School of Biomedical Sciences, The University of Queensland, St. Lucia, Queensland 4072, Australia § School of Pharmacy, The University of Queensland, Woollongabba, Queensland 4102, Australia ⊥ Institute for Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4067, Australia ‡

S Supporting Information *

ABSTRACT: Gonadotropin-releasing hormone (GnRH) agonists (e.g., triptorelin) are used for androgen suppression therapy. They possess improved stability as compared to the natural GnRH, yet they suffer from a poor pharmacokinetic profile. To address this, we used a GnRH peptide-modified dendrimer platform with and without lipidation strategy. Dendrimers were synthesized on a polylysine core and bore either native GnRH (1, 2, and 5) or lipid-modified GnRH (3 and 4). Compound 3, which bore a lipidic moiety in a branched tetramer structure, showed approximately 10-fold higher permeability and metabolic stability and 39 times higher antitumor activity against hormone-resistant prostate cancer cells (DU145) relative to triptorelin. In gonadotropin-release experiments, dendrimer 3 was shown to be the most potent construct. Dendrimer 3 showed similar luteinizing hormone (LH)-release activity to triptorelin in mice. Our findings indicate that dendrimer 3 is a promising analog with higher potency for the treatment of hormone-resistant prostate cancer than the currently available GnRH agonists.

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INTRODUCTION Gonadotropin-releasing hormone (GnRH) agonists are used to treat sex steroid-dependent carcinomas of prostate, ovaries, breast, and endometrium.1 Continuous administration of these peptide analogs act on the pituitary−gonadal axis resulting in the suppression of release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), therefore reducing testosterone and estradiol levels.2 In addition to the primary action of the GnRH receptor ligands in hormone-dependent cancers, a direct growth inhibition has been reported in hormone-unrelated cancers. GnRH receptors are overexpressed in malignant tumors such as prostate, ovarian, and pancreatic cancers, and they contribute to the antitumor activity of GnRH derivatives.3 Different studies showed antiproliferative and proapoptotic signaling in cancer cells after treatment with analogs of GnRH, particularly prostate cancer cell lines LNCaP and DU145.4−6 GnRH receptors are most abundantly overex© 2017 American Chemical Society

pressed in prostate cancer, particularly in the aggressive stages of the tumor (castration-resistant prostate cancer, CRPC).7 In 86% of prostate cancers and 80% of endometrial and ovarian adenocarcinomas, an overexpression of medium to high-affinity binding sites was observed for GnRH.8,9 While pituitary and extrapituitary GnRH-R transcripts seem identical, their functional properties have proven to be different.10 An overexpression of high affinity GnRH receptors has an important position in enhancing the direct antiproliferative effect of GnRH agonists.11 The findings justify the development of GnRH-based therapeutics for prostate cancer with direct antiproliferative action on tumor cells. Due to a very short half-life (less than 30 min), there has been extensive research into the modification of native GnRH Received: March 15, 2017 Published: September 28, 2017 8309

DOI: 10.1021/acs.jmedchem.6b01771 J. Med. Chem. 2017, 60, 8309−8320

Journal of Medicinal Chemistry

Article

Figure 1. Schematic structure of building block precursors 1′−5′ and dendrimers 1−5.

peptide to enhance its metabolic stability. GnRH analogs with better metabolic stability also had improved potency and bioavailability. It is hypothesized that this improvement resulted from longer association between the peptide analog and the GnRH receptor. Although derivatives with longer half-life than the native GnRH have been introduced to the pharmaceutical

industry (e.g., triptorelin), these GnRH derivatives still suffer from poor pharmacokinetic properties. Depot subcutaneous formulations are available; however, they can result in side effects such as leukocytoblastic vasculitis12 and injection site granulomas.13 Subcutaneous implants have also been developed but require a surgical incision that can lead to serious injection 8310



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pharmacodynamic and pharmacokinetic properties of GnRH peptide both for endocrine activity and for direct antitumor potential. We hypothesized that an improved proteolytic stability, membrane permeability, and degree of absorption from the injection site would result in a higher bioavailability and provide a sustained interaction with GnRH receptors. This is critical for the process of receptor or postreceptor desensitization for endocrine activity and would promote the activation of GnRH-receptor mediated cell-signaling pathways to induce cell death.26 Five different GnRH dendrimers (1−5; Figure 1) were synthesized using convergent (1, 2, 4, and 5)27 or divergent (3) approaches. Dendrimers were comprised of a polylysine core and GnRH in 1, 2, and 5 or lipid-modified GnRH in 3 and 4. In dendrimer 5, two C12 moieties were incorporated into the polylysine core. The pharmacological characteristics of these dendrimers were tested, including their transport through biological membranes and metabolic stability against enzymatic degradation. Direct antiproliferative activity was also examined against different GnRH receptor positive and negative tumor cell lines. The GnRH receptor mediated growth inhibitory effect, gonadotropin releasing activity (in vitro and in vivo), and the impact of steroids on the dendrimers’ antiproliferative effect was investigated. Dendrimer 3 showed outstanding activity, supporting the use of lipophilic dendrimers as a strategy to improve the stability of short peptides.

site reactions, particularly in immunocompromised cancer patients.14 Another drawback of the currently available GnRH analogs is that they are only effective in the treatment of hormone-dependent reproductive cancers. Androgen deprivation therapy (ADT), most commonly mediated by GnRH agonists,15 is the second most common treatment for prostate cancer, the most predominant type of cancer among men in Australia.16 Unfortunately, ADT is not a long-term solution: recurrence occurs in 30−50% of patients, and eventually all metastatic prostate cancer cases progress to a state of relapsing CRPC, where ADT is no longer effective. Dendrimers pose an exciting platform to create well-defined molecular structures with tailored surface functionality that is responsive to a given biological environment, mimicking the three-dimensional structure of proteins.17 Biodegradable peptide dendrimers have excellent compatibility in biological and therapeutic levels with high stability against enzymatic degradation18 and thus are attractive candidates for developing potent drug delivery systems. The multivalent presentation of bioactive ligands through dendrimeric scaffolds provides a high density of functional terminal groups, allowing the attachment of multiple active molecules.19 Dendrimers in comparison with other carriers such as, liposomes or colloids, have been shown to have enhanced biological stability against enzymatic degradation. The development of GnRH-functionalized poly(amidoamine) dendrimer (PAMAM G5) has been investigated, and the modified conjugates were reported to be stable after having been incubated for 3 days in phosphate-buffered saline (PBS) at 37 °C.20 In recent years, there has been growing attention in the utilization of biocompatible dendrimers made up of biodegradable polylysine-based structures. The biodegradable polylysine dendrimers have reduced toxicity in comparison to polyamidoamine (PAMAM) dendrimers that are the most common class of dendrimers.21 Herein we used dendritic architecture with or without lipoamino acid modification to improve the pharmacodynamic and pharmacokinetic behavior of the GnRH peptide. Lipoamino acids are α-amino acids with alkyl side-chain lengths of 8−20 carbons. The amphipathic nature of the lipoamino acids enhances the membrane permeability, metabolic stability, and bioavailability of peptides and has been extensively investigated in drug delivery.22,23 We have reported that increasing the length of the alkyl chain of lipoamino acids improved the permeability of the carrier systems across cellular membranes. However, due to solubility limitations, the 2-amino-dodecanoic acid (C12) was found to be an optimum length for drug delivery purposes.24,25 C12 was utilized in the dendrimers to further improve the resistance to enzymatic degradation and enhance the potency and bioavailability of the dendritic constructs. This will in turn provide an effective association and prolonged interaction between the GnRH dendrimers and the GnRH receptor due to an increase in the metabolic stability. Furthermore, we propose that four branches of GnRH attached to a core in a dendritic platform confers several advantages over using a 4-fold higher amount of the corresponding monomeric GnRH peptide. It provides (1) significantly improved metabolic stability against enzymatic degradation, (2) facilitated interactions with the receptors at the cell surface due to the higher number of ligands reaching the receptor at the same time, and (3) enhanced permeability through biological membranes. In this study, we took advantage of using dendrimeric structures with or without lipidation to enhance the

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RESULTS Design and Synthesis of GnRH Dendrimers. Dendrimers of GnRH were synthesized using convergent (1, 2, 4, and 5)27 and divergent (3) approaches. Building block precursors were purified by analytical reverse phase high performance liquid chromatography (RP-HPLC) (>95% purity) and characterized by electrospray ionization mass spectrometry (ESI-MS). Dendrimers 1, 2, 4, and 5 were synthesized via reverse thioether ligation yielding dendrimers with four copies of GnRH peptide conjugated to a tetrathiol dendron core.27 In dendrimers 2 and 4, Gly6 was replaced with D-Lys to allow conjugation of GnRH peptide to the core. In dendrimer 4, Leu7 was substituted by a lipid moiety, while in dendrimer 5, two copies of the lipid was attached to the Lys core. Dendrimer 3 was synthesized through a divergent Fmoc SPPS approach to yield a dendrimer with a polylysine core that bears four C-terminally lipidated GnRH peptides (Figure 1). The retention time (TR) for dendrimers 1 and 2, which do not bear any lipid moiety, was found at 14.3 and 14.4 min in analytical RP-HPLC according to the method described in Table 3. Meanwhile, the TR for lipid-bearing dendrimers 3, 4, and 5 was found to be higher, 19.6, 21, and 25.3 min, respectively. This indicates the higher lipophilicity of these analogs. Among the three lipid-modified dendrimers, dendrimer 3 was shown to be the least hydrophobic compound. Caco-2 Cell Homogenate Stability and Membrane Permeability. The metabolic stability of GnRH dendrimers was studied using a conventional colorectal adenocarcinoma (Caco-2) cell homogenate assay. All dendrimers showed increased stability with half-lives between 45 and 201 min compared to the native peptide with t1/2 of 9 min. Compounds 3 and 4 showed the highest resistance against enzymatic hydrolysis with t1/2 of 201 and 112 min, respectively (Table 1). In order to examine whether the dendrimeric structures of GnRH peptide have improved permeability, compounds 1−5 were tested in Caco-2 cell monolayer model for drug 8311



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the antiproliferative activity of the dendrimers and GnRH with IC50 > 100 μM for all compounds (Table 2). The IC50 of dendrimer 3 was shown to be significantly lower in DU145, LNCaP, and OVCAR-3 cell lines in comparison with the clinically available superagonist, triptorelin (p < 0.05, Table 2). Dendrimers were also tested against human peripheral blood mononuclear cells (PBMCs) and isolated rat pituitary cells to investigate whether they exert antiproliferative activity only against GnRH receptor positive cancer cells or they also affect normal cells. Dendrimers 1−5 and peptide controls (6 and 7) did not affect PBMC and rat pituitary cell proliferation in an MTT assay performed in the same way as for the tumor cell lines (Figure 2, p > 0.05). Impact of Steroid Reconstitution on Dendrimers’ Growth Inhibitory Effect. DU145 and OVCAR-3 cells were treated with dendrimers with or without DHT and E2, respectively. Using CSS media that contained serum depleted of low-molecular-weight lipophilic compounds such as steroid hormones resulted in a 26%, 81%, 27%, and 34% reduction (p < 0.05) in the sensitivity of DU145 cells to dendrimers 2, 3, and 4 and triptorelin, respectively (p < 0.05, Figure 3a). However, this effect was restored after addition of DHT to the media. Although the proliferation of OVCAR-3 cells decreased by 23%, 42%, and 16% (2, 3, and 4, respectively; p < 0.05) in CSS media, reconstitution of the growth media with E2 fully overcame this effect (p < 0.05, Figure 3b). Triptorelin Competition Studies. DU145, LNCaP, and OVCAR-3 were pretreated with 100 μM triptorelin for 2 h. The significant antiproliferative effects of dendrimers 2 and 3 (in all 3 cell lines) and 4 (in DU145 and OVCAR-3) was reversed after pretreatment with triptorelin. The effect was reduced by 20−40% in different cells lines by dendrimers 2−4 (Figure 4a− c). Although an IC50 of 48 μM was achieved for dendrimer 3 in the competition study, its growth inhibitory effect was significantly reduced in pretreated experimental conditions compared to the normal conditions (p < 0.05). In Vitro LH and FSH Release Study. The effect of the dendrimers on LH and FSH release was examined in vitro by incubating rat pituitary cell cultures with the dendrimers for 2 h. Dendrimer 3, GnRH and triptorelin significantly increased the FSH release at all three concentrations (0.5, 5, and 50 nM) from 1.5 to 2 ng/mL compared to the negative control (1.1 ng/ mL, p < 0.05, Figure 5a). Dendrimer 4 also stimulated the release of FSH to 1.6 ng/mL at 0.5 nM. The other three dendrimers did not affect the FSH release in the cultured pituitary cells (p > 0.05, Figure 5a). Dendrimers 2 (at 5 and 50 nM) and 4 (at 50 nM) increased the level of LH up to 67 ng/mL (p < 0.05, Figure 5b). Similar to its effect on FSH, dendrimer 3 stimulated the LH release at 0.5, 5, and 50 nM to 69, 91, and 83 ng/mL, respectively (p
100 87.9 ± 8.1 >100 >100 >100

76.2 ± 6.2 81.4 ± 5.3 >100 66.3 ± 5.9* >100

1.6 ± 0.4***,# 41.6 ± 3.5*,# 80.1 ± 8.8* 21.5 ± 3.1*,# >100

66.4 ± 0.9* >100 >100 52.1 ± 5.5* >100

>100 >100 >100 >100 >100

87.7 ± 9.9 88.6 ± 6.7 >100 90.1 ± 3.4 >100

triptorelin (7) 62.1 ± 73.4 ± 98.1 ± 67.7 ± >100

6.2 6.3 6.4 4.4

a The IC50 values (μM) were estimated from concentration−response curves using nonlinear regression for inhibition of cell growth. Data are expressed as mean ± SEM from at least three independent experiments (n = 3). Statistical analysis was performed using a two-way ANOVA (*p < 0.05 and ***p < 0.0001 for the IC50 for each dendrimer when compared with GnRH, and #p < 0.05 when compared with triptorelin).

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Figure 2. Toxicity of dendrimers 1−5 in (a) PBMCs and (b) isolated rat pituitary cells as a percentage of cells treated with PBS (mean ± SEM, performed in three independent experiments, each in triplicate). Statistical analysis was performed using a one-way ANOVA followed by Dunnett’s post hoc test and comparison with the DMSO group.

response for triptorelin was obtained after 2.5 h at 1.40 ng/mL. However, the LH release stimulated by dendrimer 3 continued to increase to 1.43 ng/mL after 3 h (Figure 6a). The area under curve (AUC) of LH release was also obtained to provide an indication of the duration of action. A significant increase was observed in the AUC of triptorelin (1.3 ng/(mL·6 h)) and dendrimer 3 (1.5 ng/(mL·6 h)) when compared with PBS control group of mice (p < 0.05, Figure 6b). The AUC calculated for dendrimer 3 was slightly higher than that of triptorelin.

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DISCUSSION Significant growth inhibitory effects of clinically available GnRH agonists (such as triptorelin) against different cancer cells have been reported in preclinical studies.4−6 This is an added benefit in the treatment of hormone-sensitive tumors; unfortunately, the effect is not strong enough to make these agonists appropriate for the treatment of hormone-refractory tumors. In this study, dendrimer 3 had approximately 10 times better metabolic stability and permeability and produced a 39fold higher antiproliferative effect in androgen-resistant DU145 prostate cancer cells than triptorelin. The antitumor activity was directly dependent on the lipophilicity of the compounds.28 This may have resulted from improved interaction with the cell membrane and better drug uptake, resulting in higher bioavailability, longer duration of action, and improved tumor penetration.29 We showed that the attachment of a lipidic moiety to the C-terminus of GnRH in a branched tetramer structure, 3, not only exhibited an enhanced membrane permeability and improved metabolic stability of the GnRH peptide but also induced up to 55 and 39 times higher antitumor activity relative to the parent peptide and triptorelin, respectively. The results of the triptorelin competitive assay suggested that the growth inhibitory effect of dendrimer 3 was mediated via the GnRH receptor. Several studies have revealed that modifications of the backbone structure of GnRH peptide could significantly improve stability. This in turn enhanced the pharmacological activity despite a potential reduction in the receptor binding affinity. Amide-linked cyclic GnRH analogs had significantly improved in vitro and in vivo stability and enhanced pharmacokinetics properties compared with their linear counterparts.30 Another bioactive analog of GnRH, [DesGly10,Tyr5(OMe),D-Leu6,Aze-NHEt9]GnRH, was considerably more stable in vitro and in vivo as compared to leuprolide. This compound induced testosterone release in acute administration to mice with similar antiproliferative activity as

Figure 3. Effect of DHT and E2 reconstitution on the sensitivity of (a) DU145 and (b) OVCAR-3 cells to the antiproliferative activity of dendrimers. For the first 48 h, cells were grown in steroid free (CSS) media. Cells were either reconstituted with E2 (5 nM) or DHT (50 nM) or treated with dendrimers at 50 μM. Reconstituted cells were treated with compounds 1−6 for a further 48 h. *p < 0.05 experiments performed in normal media vs CSS media. #p < 0.05 experiments performed in steroid reconstituted media in comparison with CSS and normal media.

0.05). GnRH and triptorelin (at 50 nM) also boosted the level of LH to 69 and 87 ng/mL, respectively, compared to the negative control (46 ng/mL; p < 0.05). Dendrimers 1 and 5 were not observed to affect LH release (p > 0.05, Figure 5b). The Effect of the Dendrimers on LH Release in Mice. Dendrimer 3 was selected because of its results in in vitro studies; this analog showed the highest stability and efficacy in inhibiting the growth of cancer cells and release of gonadotropins. Dendrimer 3 and triptorelin (50 μmol in 50 μL/mouse) were administered subcutaneously to the mice, and LH release was measured every 30 min for 6 h. Both compounds stimulated the gradual release of LH. The peak 8313



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Figure 5. Effect of GnRH dendrimers on the release of (a) FSH and (b) LH after incubation with rat pituitary cells. Statistical analysis was performed using a one-way ANOVA followed by the Dunnett’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001; increase in the LH level in comparison with the PBS control).

gonadotropins in both cultured pituitary cells and mice. Results from in vitro and in vivo efficacy studies suggested that effective GnRH receptor-mediated pathways were still activated after Cterminal lipidation of GnRH and assembly of the peptide analog on a polylysine core. It is also plausible that the enhancement in the antiproliferative potential of 3 compared to triptorelin and native peptide is due to a highly stabilized conformation that supports attachment to the overexpressed GnRH receptors. Dendrimer 3 was approximately 2 and 3 times more potent than triptorelin in inhibiting the growth of hormone-sensitive cell lines LNCaP and OVCAR-3, respectively. Consistent with earlier research using triptorelin or other GnRH analogs,33 dendrimers 1−5 did not inhibit the growth of SKOV-3 cells with lower levels of GnRH receptors.5 Dendrimers 1, 2, and 4 shared the same tetrathiol dendron core structure. Dendrimers 2 and 4 were conjugated to the core through the middle of the GnRH sequence, while dendrimer 1 was attached to the C-terminus of the GnRH peptide. Dendrimer 4 exhibited significantly higher metabolic stability and membrane permeability and slightly better antiproliferative and gonadotropin-release efficacy in vitro than dendrimer 2, possibly due to the additional midsequence lipidation. However, dendrimer 4 was still less effective than dendrimer 3 at inhibiting the growth of tumor cells and stimulating the

Figure 4. Receptor-mediated antiproliferative activity of dendrimers in (a) DU145, (b) LNCaP, and (c) OVCAR-3 cells. Cells were pretreated with 100 μM triptorelin for 2 h, and then the supernatant was replaced with fresh media containing compounds 1−8 and control peptides at 50 μM. MTT assay was performed after 48 h incubation at 37 °C in a 5% CO2 atmosphere (mean ± SEM, performed in three independent experiments, each in triplicate). Statistical analysis was performed using a one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, comparison was made between pretreated and untreated groups. DMSO 1% is included to show that the vehicle did not have any significant impact on the cell proliferation).

leuprolide on androgen-dependent prostate cancer (LNCaP) cells.31 An autocrine/paracrine GnRH loop in androgen-independent prostate cancer cells (e.g., DU145) was previously suggested to regulate tumor cell growth.32 Dendrimer 3 also exhibited significant efficacy in stimulating the release of 8314



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Figure 6. Dendrimer 3 (50 μmol/mouse) evoked release of LH after SC administration in mice. (a) LH level in plasma was measured using a sensitive micro-ELISA method during the first 6 h post-treatment and (b) the AUC over the 6 h sampling period following administration of dendrimer 3, triptorelin (50 μmol/mouse), and PBS (mean ± SEM (n = 5)). Statistical analysis was performed using a one-way ANOVA followed by the Dunnett’s post hoc test. (***p < 0.001; dendrimer 3 and triptorelin treated groups compared to PBS control group).

growth inhibitory effect of a GnRH agonist. A down-regulation of GnRH receptor mRNA was responsible for the attenuation of growth inhibition after treatment with E2.36 In gonadotropin-release efficacy studies, dendrimer 3 was the most potent construct, with comparable potency to triptorelin in stimulating the release of FSH and LH. Dendrimer 3 showed similar LH-release activity to triptorelin in mice. This effect indicated that the compound has effective binding affinity for the GnRH receptors and served as a GnRH receptor agonist, which produced an initial intense stimulation of the gonadotrophic cells to release LH. Continuous pituitary overstimulation lead to down-regulation/desensitization of GnRH receptors, which consequently decreased sex hormone levels, resulting in “chemical castration”.38 Considering its significantly higher antiproliferative activity than triptorelin, dendrimer 3 is a promising analog with superior potency for the treatment of hormone-refractory prostate cancer and warrants further investigation.

release of FSH and LH. Despite significant improvement in the metabolic stability and permeability, the antiproliferative activity and ability to stimulate the release of gonadotropins was not superior to triptorelin; thus dendrimer 4 was not tested in mice. The decreased biological efficacy of 1, 2, and 4 was partly explained by the presence of a bulky tetrathiol dendron core, which might have compromised effective receptor binding. The negative impact of a bulky core was even more prominent in dendrimer 5 where two lipidic moieties (C12) were attached to the tetrathiol dendron structure and completely abolished its biological activity. Collectively, lipid modification increased stability, permeability, and biological activity only when the lipid was directly conjugated to the GnRH peptide (3 and 4) and not to the core (5). The antiproliferative effects of dendrimers were specific to cancer cells that overexpressed the GnRH receptors and did not adversely affect the growth of PBMC and pituitary cells. This was in agreement with a previous report by Miles et al. who did not observe any significant pro-apoptosis induction by GnRH in LβT2 gonadotrope cells.34 Steroid hormones were previously shown to affect the growth inhibitory effect of GnRH agonists.35,36 We observed a significant reduction in the sensitivity of DU145 cells to the antiproliferative effects of dendrimers and triptorelin in CSS media. The restoration of this effect after cotreatment with DHT suggested that the growth inhibitory effects of dendrimers 2−4 were steroid-dependent. These results were in agreement with previous reports on the steroid-dependent antiproliferative effects of GnRH agonists.35 A 119% DHTdependent upregulation of membrane GnRH receptor expression in GnRH-receptor positive prostate cancer cells was responsible for the increased activity of GnRH agonists.37 The higher sensitivity of DU145 cells to GnRH dendrimers could be the result of the increased number of GnRH receptors in the presence of DHT. In OVCAR-3 cells, however, the antiproliferative effects of dendrimers 2−4 were unaffected in the absence of hormones (CSS media). In E2-reconstituted media, the sensitivity of the OVCAR-3 cells to dendrimers 2−4 significantly decreased compared to the CSS media. This observation could be explained by the mitogenic activity of estrogen in OVCAR-3 cells, which could antagonize the growth inhibitory effect of GnRH dendrimers.36 This was in agreement with a previous study where pre- or cotreatment with E2 greatly attenuated the

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CONCLUSION We successfully developed some new GnRH dendrimeric structures that not only possess significant potential to cause chemical castration but also produced direct tumor growth inhibitory activity in hormone-refractory prostate cancer cells. This significant finding provides a promising new therapy to treat prostate cancer. Furthermore, these dendrimers can be used as model structures for the development of new GnRH agonists with dual endocrine and antitumor activities.

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EXPERIMENTAL SECTION

General. For peptide synthesis, protected L-amino acids and resins were supplied by Novabiochem (Läufelfingen, Switzerland), Reanal (Budapest, Hungary), or Mimotopes (Melbourne, Australia). Peptide synthesis grade DMF was purchased from Labscan (Dublin, Ireland). A 0.45 μM (47 mm) nylon filter was used to filter DMF before each use. HBTU was supplied by Mimotopes (Melbourne, Australia), and TFA and DIPEA were procured from Merck KGaA (Darmstadt, Germany). Analytical grade acetonitrile (MeCN) was purchased from Scharlau (Barcelona, Spain) or Labscan (Dublin, Ireland). Other chemicals were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia) at the highest purity unless otherwise stated. Biological reagents were primarily acquired from Gibco-BRL (Grand Island, NY). HBSS, HEPES, and propanolol were sourced from SigmaAldrich (Castle Hill, NSW, Australia) and 14C-mannitol from Amersham Biosciences (Piscataway, NJ). Tissue culture flasks (TPP1 75 cm2) and normal 96-well plates were ordered from Becton 8315



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Table 3. Characterization of Building Blocks (1′−5′) and Dendrimers (1−5) RP-HPLC ESI-MS building block precursors

MW

2′

[(Mpa)2K-C12]2KRS-NH2

1391.96

5′

pEHWSY[K-BrAc]C12RPG-NH2

1458.5

analytical method

TR (min)

method

50%−70%B in 30 min, C18 column

20.1



17.2


detected (m/z) 1392.6 [M]+ 696.8 [M]2+ 1460.7 [M]+ 730.3 [M]2+

preparative

RP-HPLC ESI-MS dendritic constructsa

MW

1

[((pEHWSYGLRPGK)-Mpa)2K]2K-NH2

6155.99

2

[((pEHWSYKLRPG)-Mpa)2K]2K-NH2

5927.79

3

[(pEHWSYGLRPG-C12)2K]2K-NH2

5851.85

4

[((pEHWSYK-C12-RPG)-Mpa)2K]2K-NH2

6264.43

5

[(pEHWSYGLRPGK-Mpa)2K-C12]2KRS-NH2

6793.89

analytical

detected (m/z) 1540.6 [M]4+ 1232.4 [M]5+ 1027.7 [M]6+ 880.9 [M]7+ 771.1 [M]8+ 1484.2 [M]4+ 1187.3 [M]5+ 989.5 [M]6+ 848.1 [M]7+ 743 [M]8+ 1464.9 [M]4+ 1172.2 [M]5+ 976.8 [M]6+ 838 [M]7+ 1568.1 [M]4+ 1255 [M]5+ 1045.8 [M]6+ 896.5 [M]7+ 784.8 [M]8+ 1360.2 [M]5+ 1134.5 [M]6+ 972.2 [M]7+ 850.8 [M]8+ 756.4 [M]9+

method

preparative TR (min)

method


14.3



14.4



19.6


30%−45%B in 30 min,C4 column

21



25.3


a

Amino acid sequences of minidendrimeric GnRH derivatives, dendrimers 1 (1′ + 3′), 2 (1′ + 4′), 3 (divergent approach), 4 (1′ + 5′), and 5 (2′ + 3′). Dickinson; high-affinity microplates (9018) were obtained from Corning (USA). Transwell polycarbonate inserts (mean pore size 1/ 4 0.45 μm, 6.5 mm diameter) were purchased from Costar (Cambridge, MA). Charcoal Stripped Fetal Bovine Serum was sourced from Life Technologies (Gibco). Monoclonal antibody, anti-bovine LH beta subunit (51B7), was sourced from the University of California, USA, polyclonal antibody, rabbit LH antiserum (AFP240580Rb) from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), The National Hormone and Peptide Program (NHPP), USA, polyclonal goat anti-rabbit from DakoCytomation, Australia, and o-phenylenediamine (002003) from Invitrogen, Australia. Absorbance measurements were conducted on a Varian Cary 50 Bio UV/vis spectrophotometer (λ = 570 nm). Transepithelial electrical resistance (TEER) was measured using the Millicell-ERS epithelial

volt-ohmmeter system (Millipore Corporation). Analytical reverse phase high performance liquid chromatography (RP-HPLC) was carried out using a Shimadzu instrument (Kyoto, Japan) fitted with a SIL-20AC HT autosampler, LC-20AB (binary) pumps with a flow rate of 1 mL/min, and a UV detector set to a wavelength of 214 nm. Preparative RP-HPLC was performed using a Shimadzu system (Kyoto, Japan; LC-20AT, CBM-20AV, SPD-20AV, FRC-10A) with a flow rate of 20 mL/min and UV detection at 214 nm. The mobile phases for both analytical and preparative RP-HPLC were solvent A (0.1% TFA in water) and solvent B (0.1% TFA in water-MeCN, 1:9, v/v). Analytical separation was achieved by applying a linear gradient of solvent B over 30 min on either Vydac C18 (4.6 mm × 250 mm, 5 μm) or C4 (4.6 mm × 250 mm, 5 μm) to determine a purity of ≥95% for all building blocks and final products, while preparative separation used Vydac C18 (22 mm × 250 mm, 10 μm) or C4 (22 mm × 250 8316



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mm, 10 μm). Electrospray ionization mass spectrometry (ESI-MS) was performed on a PE Sciex API3000 triple quadrupole mass spectrometer using a mixture of solvent A (0.1% formic acid in water) and B (0.1% formic acid in 9:1 acetonitrile/water) at 0.05 mL/min. Liquid chromatography−mass spectrometry (LC-MS) was carried out using solvents A and B at a flow rate of 0.3 mL/min; a splitter after the Phenomenex Luna C18 column (5 μm, 50 mm × 2.0 mm) was used to achieve a flow rate of 0.05 mL/min in the ion source of the mass spectrometer. Preparative RP-HPLC was performed on a Waters 600 controller and pump with a 490E programmable multiwavelength UV/ visible detector, and analytical RP-HPLC was carried out on an Agilent 1100 system fitted with a binary pump, autosampler, and UV detector set to a wavelength of 214 nm. Chemistry: Synthesis and Purification of GnRH Derivative Minidendrimers. Tetrathiol polylysine dendron core peptides (compounds 1′ and 2′; 100−200 μmol scale) and GnRH peptide derivatives (compounds 3′, 4′, and 5′; 200−400 μmol scale) were used as the starting materials for further synthesis of GnRHfunctionalized dendrimers (compounds 1, 2, 4, and 5, at 4 μmol scale). These compounds were synthesized by a convergent approach on Rink amide MBHA resin using microwave-assisted Fmoc solidphase peptide synthesis (SPPS) according to a previously published method.27 Dendrimer 3 was synthesized using a divergent approach on Rink amide MBHA resin using microwave-assisted Fmoc-SPPS procedure (25 μmol scale). The synthesis and purification of peptides 1′, 3′, and 4′ and dendritic constructs (compounds 1 and 2) was performed according to previously published procedures.27 Core peptide 2′ contained C12 lipoamino acid ([(Mpa)2KC12]2KRS-NH2) and was synthesized by introducing Fmoc-Lys(Fmoc)-OH as a branching unit (Figure 1), followed by addition of Dde-D12-OH and functionalization with Trt-Mpa (8 equiv; 2 equiv per branch of the tetravalent dendron core). Amino acids were preactivated by HATU (8 equiv)/DIPEA (10 equiv). To synthesize compound 5′, Dde-C12-OH lipoamino acid was coupled to position 7 of the GnRH sequence followed by addition of Fmoc-D-Lys(Dde)-OH at position 6. The bromoacetyl functionalization of peptide 5′ was achieved after subsequent Dde deprotection of the Fmoc-D-Lys(Dde)OH, followed by treatment with a mixture of bromoacetic acid (10 equiv) and N,N-diisopropylcarbodiimide (5 equiv). Compounds 2′, 5′, and 3 were then cleaved from the resin using a cocktail of TFA/water/ triisopropylsilane (TIS)/1,2-ethanedithiol (EDT) (80:5:5:10) (2′) or TFA/water/TIS (95:2.5:2.5) (5′ and 3) over 4−8 h. Crude products 2′, 5′, and 3 were purified by preparative RP-HPLC, and the fractions were analyzed by ESI-MS and analytical RP-HPLC (Table 3). Pure fractions were collected together, lyophilized, and kept at −20 °C. Dendrimers 4 and 5 were synthesized via a convergent thioether ligation approach (4 μmol scale) from their pure precursors (>95% on analytical RP-HPLC): tetrathiol dendron core (1′ and 2′ for constructs 4 and 5, respectively) and bromoacetyl-functionalized GnRH derivatives (5′ and 3′ for constructs 4 and 5, respectively). Dendron cores (4 μmol) were dissolved in 2 mL of aqueous sodium bicarbonate solution (20 mM, pH 7.5) containing tris(2carboxyethyl)phosphine (TCEP, 0.5 equiv per branch of the core; 8 μmol). The bromoacetylated GnRH derivatives (2 equiv per branch of the core; 32 μmol) dissolved in sodium bicarbonate solution (18 mL, 20 mM, pH 7.5) were then added to the reduced dendron core. The ligation reactions were monitored using analytical RP-HPLC on a C4 column using a gradient of 30%−40% solvent B over 30 min (for dendrimer 4) or 22%−37% of solvent B over 30 min (for dendrimer 5). The ligation reactions between precursors were completed after 3 h, and the mixtures were then injected into the preparative RP-HPLC using the methods listed in Table 3. The collected fractions were analyzed using ESI-MS and analytical RP-HPLC (Table 3). The pure fractions were collected together, lyophilized, and stored at −20 °C. Cell Culture. The Caco-2 human epithelial colorectal adenocarcinoma line was supplied by the American Type Culture Collection (Rockville, MD, USA). Caco-2 cells were cultured in 75 cm2 flasks (Nunc, Australia) with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% nonessential amino acids at 95% humidity at 37 °C under an

atmosphere of 5% CO2. The medium was replaced by the fresh culture medium every other day. Cells were subcultured at 80% confluency using 0.25% trypsin−EDTA, and the cells with passage numbers of 35−49 were used in the assays. Different steroid hormone-dependent and -independent, sexspecific human carcinoma cell lines were used. The LNCaP (GnRH receptor positive; androgen-sensitive prostate adenocarcinoma), PC3 and DU145 (GnRH receptor positive; androgen-independent human carcinoma), OVCAR-3 (GnRH receptor positive; steroid hormonesensitive ovarian carcinoma), and SKOV-3 (GnRH receptor negative; estrogen-resistant ovarian carcinoma) human cell lines were also used and were kindly provided by Professor Judith Clements at the Translational Research Institute, Queensland University of Technology, and Professor Rodney Minchin, School of Biomedical Sciences, The University of Queensland. Cells were maintained in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS) and PenStrep at 37 °C in a 5% CO2 atmosphere. Caco-2 Cell Homogenate Stability Assay. Caco-2 cell stability assay was performed according to previously published procedures.39 Briefly, Caco-2 cells were cultured in a 75 cm2 flask. After about 2 weeks, when the flask was 80% confluent, cells were trypsinized and resuspended in Hank’s Balanced Salt Solution containing 25 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HBSS-HEPES, pH 7.4). The cell homogenate was prepared using a probe sonicator (Sonics Vibracell ultrasonic processor, Sonic and Materials Inc., Danbury, CN). Two times 1 s pulses were applied at 60% amplitude at 130 W to lyse the membranes followed by centrifugation (2000 rpm, 5 min) and protein content determination using the Bio-Rad protein assay kit (0.6−0.9 mg/mL). The peptides (200 μM, 100 μL) were incubated with the cells at 37 °C in a shaker at 400 rpm, and samples (10 μL) were collected at preselected time points (5, 10, 15, 20, 30, 40, 50, 60, and 120 min). At the end of the experiment, 5 μL of TFA was added to stop the enzymatic digestion. Diluted samples were analyzed by LC-MS, and the concentration of the intact peptide was determined. LC-MS was performed on a Luna Phenomenex C18 column. The mass spectrometer was set to selective ion monitoring in the positive ion electrospray mode. The concentration of the peptide in the samples was determined based on the standard curve plotted from standards with known peptide concentrations. Analyst software version 1.4.1 was used for instrument control, data acquisition, and data analysis. Caco-2 Cell membrane Permeability Assay. Permeability of the dendrimers across biological membranes was assessed according to a published method.39 Briefly, a suspension of Caco-2 cells (1 × 106 cells/mL, 100 μL) was pipetted into polycarbonate cell culture inserts (pore size 0.4 μm, 6.5 mm diameter, Transwell) in a 24-well plate and 0.6 mL aliquots of medium were added to the basolateral chamber. The culture medium was changed every second day. The integrity of the tight junctions and the monolayers was assessed by measuring the transepithelial electrical resistance (TEER) values. The TEER values of the monolayers were between 1700 and 4000 ± 500 Ω cm2 with no significant drop in the values after the completion of the experiment, indicating that the compounds were nontoxic to the cells. Radiolabeled [14C]-D-mannitol was used to monitor the Caco-2 cell monolayer integrity (Sigma; 0.09 mCi/mL in 90% EtOH in water). A [14C]-Dmannitol solution (1.80 μCi, 32.73 nmol/4 mL) was added to the apical chamber of three wells. After 21−28 days, the Caco-2 cell monolayers were washed with prewarmed HBSS-HEPES, pH 7.4, and incubated for 30 min. Test and reference compounds were dissolved in HBSS-HEPES buffer and DMSO to a final concentration of 200 μM in 1% DMSO. Buffer was replaced with 100 μL aliquots of each dendrimer solution in the apical chambers, and the plates were incubated at 37 °C in a shaker at 400 rpm. Samples (0.4 mL) were collected from the basolateral chambers at selected time points 30, 90, 120, and 150 min and replaced with the same volume of buffer. The concentration of the peptide in each sample was determined by LC-MS analysis in the same method as described for the metabolic stability. The radioactivity of the [14C]-D-mannitol samples was quantified by liquid scintillation counting (Liquid Scintillation 8317



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Systems, BECKMAN LS3801, USA). The Papp for each compound was calculated from the data using the following equation:

media was replaced by challenge media that contained DMEM with 0.1% BSA. The cells were incubated at 37 °C for 2 h with the GnRH dendrimers or control peptides (GnRH and triptorelin) at 0.5, 5, and 50 nM (10 μL). The level of LH and FSH gonadotropins was quantified using a commercial ELISA kit according to the manufacturer’s instructions (Uscn Life Science Inc., Wuhan, China). In Vivo Experiments. Approval was obtained from The University of Queensland Animal Ethics Committee (AEC#SCMB/005/11/ ARC) to perform the animal experiments according to NHMRC animal handling guidelines. Male Swiss Albino mice (6 mice/group, 6−8 weeks of age, weighing 34−45 g at the time of assessment) were obtained from The University of Queensland biological resources breeding facility (UQBR). Mice were housed in groups of five per cage in an artificially lit room on a 12 h light/12 h dark cycle at controlled temperature (22.2 ± 0.2 °C; mean ± SEM) and humidity (51−65%) with free access to food and water. Mice were acclimatized to the new environment for 2 weeks before commencing the experiments. In order to minimize stress, better control LH pulsatility, and thus reduce experimental variation, mice were trained every alternative day by mimicking experimental manipulation procedures. Dendrimers were dissolved in 10% DMSO and administered (50 μmol in 50 μL) subcutaneously. Baseline blood samples were collected 1 h prior to administering the compounds via a single tail tip blood sample adhering to guidelines established previously.41 Blood samples were collected (2 μL) at 30 min intervals after administration for 6 h. The collected blood was diluted in 58 μL of PBS-T and immediately placed on dry ice to avoid decomposition of LH. Samples were kept at −80 °C prior to batch analysis using a validated ultrasensitive mouse LH ELISA method.41 Assessment of in Vivo LH Secretion. A sensitive sandwich ELISA was used to measure serum concentration of LH, strictly adhering to published methodology.41 Briefly, 96-well high-affinity binding microplates were coated with anti-bovine LH beta subunit monoclonal antibody (50 μL, University of California) and incubated overnight at 4 °C. A 2-fold serial dilution of mouse LH (1.95 × 10−3 to 4 ng/mL) was prepared in 0.2% (w/v) BSA-1 × PBS-T (PBS with 0.05% Tween 20), to generate a standard curve. Wells were incubated with 200 μL of blocking buffer (5% skim milk in PBS-T) for 2 h at room temperature (RT). The LH standards were incubated with detection antibody (polyclonal antibody, rabbit LH antiserum, 50 μL) and plasma samples for 1.5 h at a final dilution of 1:(1 × 105) at RT. Horseradish peroxidase-conjugated antibody (50 μL, polyclonal goat anti-rabbit antibody) was then added at a final dilution of 1:(2 × 104) and 1.5 h incubation at RT. This step was followed by adding Ophenylenediamine substrate (containing 0.1% H2O2) and incubating at RT for 30 min. Hydrochloric acid (3 M) was used to stop the reaction. The absorbance of each well was read at a wavelength of 490 nm (Sunrise; Tecan Group). The LH concentrations were determined from interpolated OD values against a nonlinear regression of the LH standard curve. LH secretory responses were also expressed as the area under the curve (AUC). A within and between assay coefficient of variation of below 5% was obtained for LH assays. Statistical Analysis. Data are presented as means ± SEM or means ± SD (LC-MS analysis). In in vitro studies, n = 3/compound, in at least 3 independent experiments. In in vivo LH-release mice studies, six mice were used for each group of test/control compounds. Differences between groups were identified by a one-way ANOVA followed by Dunnett’s post hoc test and comparison with the corresponding control group. GraphPad Prism (version 6.0; GraphPad Software Inc.) was used for all analysis. The threshold level for statistical significance was set at p < 0.05.

Papp (cm/s) = dC /dt × Vr /(A × C0) where dC/dt is the steady-state rate of change in the test item concentration (M/s) or radiochemical concentration (dpm mL/s) in the receiver (basolateral) chamber, Vr is the volume of the receiver (basolateral) chamber (mL), A is the surface area of the cell monolayers, and C0 is the initial concentration in the donor chamber (M or dpm/mL). MTT Cell Proliferation Assay. Cells were trypsinized and seeded at 90 μL/well into a flat-bottom 96-well plate (LNCaP, DU145, or OVCAR-3 cells at 2 × 105 cells/mL, PC3 and SKOV-3 cells at 1 × 105 cells/mL, and rat pituitary cells at 4 × 105 cells/mL). After 1 h incubation, compounds dissolved in 10% DMSO/PBS were added to each well (10 μL). Compounds were used at a final concentration of 1, 10, 25, 50, and 100 μM in 1% DMSO in the culture media (n = 3/ compound, in at least 3 independent experiments). MTT (5 mg/mL) was added after 48 h incubation (10 μL) and plates were further incubated for 4 h. Acidified isopropanol (100 μL, 0.1 N HCl) was used to dissolve the formazan crystals followed by pipetting and sonication in bath sonicator. The absorbance of each well was measured at 570 nm by a Spectramax 250 microplate reader. The percentage of cell viability for each compound was calculated based on the absorbance of PBS treated negative control group. SDS was used as a positive control and DMSO at a final concentration of 0.5% as a negative control. Steroid Treatment Studies. Cells were grown in media that contained 10% charcoal stripped FBS (CSS) for 48 h until they reached 70% confluence. After 48 h, cells either were seeded in 96-well plates to perform MTT assay or were treated with fresh CSS media reconstituted with 5 nM 17β-estradiol (E2) or 50 nM dihydrotestosterone (DHT). MTT assay was performed on cells grown in steroid reconstituted media. In both MTT assays, cells were treated with 50 μM of each dendrimer or control peptides. Triptorelin Competition Assay. The LNCaP, DU145, and OVCAR-3 cells were pretreated by incubating the cells with 100 μM (100 μL) triptorelin for 2 h. Cells were centrifuged for 5 min at 400g, and the media was replaced with 90 μL of fresh media. Compounds were added to each well at 50 μM (10 μL) and incubated for 48 h. An MTT assay was performed after the 48 h of incubation. Isolation of Peripheral Blood Mononuclear Cells (PBMCs). This assay was performed following approval from the University of Queensland Ethics Committee (Ethics Approval Number: 2009000661). PBMCs were isolated from whole blood (4 mL) taken from a healthy adult volunteer and prepared on Ficoll after centrifugation at 400g for 30 min. The mononuclear cell layer formed at the plasma−Ficoll interface was collected without disturbing the plasma−Ficoll interface and washed with RPMI 1640 (×3). Cells were counted and seeded at 1 × 106 cells/mL (80 μL) in a 96-well flat bottom plates along with phytohemagglutinin (10 μg/mL) in order to activate them during incubation time. After 1 h incubation at 37 °C, compounds were added (25 and 50 μM) in 10% DMSO/PBS (10 μL/ well). An MTT assay was performed after 48 h incubation. Rat Pituitary Cell Preparation. Pituitary cells were isolated and cultured according to a previously published method.40 Briefly, rats (male, Sprague−Dawley, 6−8 weeks, 120−180 g, purchased from The University of Queensland biological resources breeding facility (UQBR)) were euthanized by CO2 inhalation, then the anterior pituitaries were removed immediately, rinsed with HBSS-HEPES (25 mM, pH 7.2), and minced into small pieces using a razor blade. Pituitary fragments were incubated in a collagenase enzyme solution (1 mg/mL dissolved in 1% bovine serum albumin) for 1 h at 37 °C. Using a cell strainer (Costar), cell clumps were removed. The suspension was centrifuged at 400g for 10 min. After the supernatant was decanted, cells were resuspended in 10% FBS in DMEM media. Cells were plated in flat bottom 96-well plates at a density of 3 × 105 cells/well and incubated at 37 °C for 72 h. In Vitro Gonadotropin Release Assay. Plates that contained cultured pituitary cells were centrifuged at 1200g for 10 min. Cell

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01771. Molecular formula strings (CSV) 8318



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(5) Morgan, K.; Stewart, A. J.; Miller, N.; Mullen, P.; Muir, M.; Dodds, M.; Medda, F.; Harrison, D.; Langdon, S.; Millar, R. P. Gonadotropin-releasing hormone receptor levels and cell context affect tumor cell responses to agonist in vitro and in vivo. Cancer Res. 2008, 68, 6331−6340. (6) Dondi, D.; Limonta, P.; Moretti, R. M.; Marelli, M. M.; Garattini, E.; Motta, M. Antiproliferative effects of luteinizing hormone-releasing hormone (lhrh) agonists on human androgen-independent prostate cancer cell line du 145: Evidence for an autocrine-inhibitory lhrh loop. Cancer Res. 1994, 54, 4091−4095. (7) Moretti, R. M.; Marelli, M. M.; Taylor, D. M.; Martini, P. G. V.; Marzagalli, M.; Limonta, P. Gonadotropin-releasing hormone agonists sensitize, and resensitize, prostate cancer cells to docetaxel in a p53dependent manner. PLoS One 2014, 9 (4), e93713. (8) Halmos, G.; Arencibia, J. M.; Schally, A. V.; Davis, R.; Bostwick, D. G. High incidence of receptors for luteinizing hormone-releasing hormone (lhrh) and lhrh receptor gene expression in human prostate cancers. J. Urol. 2000, 163, 623−629. (9) Grundker, C.; Volker, P.; Emons, G. Antiproliferative signaling of luteinizing hormone-releasing hormone in human endometrial and ovarian cancer cells through g protein alpha(i)-mediated activation of phosphotyrosine phosphatase. Endocrinology 2001, 142, 2369−2380. (10) Eidne, K. A.; Flanagan, C. A.; Harris, N. S.; Millar, R. P. Gonadotropin-releasing-hormone (gnrh)-binding sites in humanbreast cancer cell-lines and inhibitory effects of gnrh antagonists. J. Clin. Endocrinol. Metab. 1987, 64, 425−432. (11) Everest, H. M.; Hislop, J. N.; Harding, T.; Uney, J. B.; Flynn, A.; Millar, R. P.; McArdle, C. A. Signaling and antiproliferative effects mediated by gnrh receptors after expression in breast cancer cells using recombinant adenovirus. Endocrinology 2001, 142, 4663−4672. (12) Gerceker Turk, B.; Dereli, T.; Dereli, D.; Akalin, T. Leuprolide acetate-induced leukocytoclastic vasculitis. Acta Obstet. Gynecol. Scand. 2007, 86, 892−893. (13) Shiota, M.; Tokuda, N.; Kanou, T.; Yamasaki, H. Incidence rate of injection-site granulomas resulting from the administration of luteinizing hormone-releasing hormone analogues for the treatment of prostatic cancer. Yonsei Med. J. 2007, 48, 421−424. (14) Abouelfadel, Z.; Crawford, E. D. Leuprorelin depot injection: Patient considerations in the management of prostatic cancer. Ther. Clin. Risk Manage. 2008, 4, 513−526. (15) Shore, N. D.; Abrahamsson, P. A.; Anderson, J.; Crawford, E. D.; Lange, P. New considerations for adt in advanced prostate cancer and the emerging role of gnrh antagonists. Prostate Cancer Prostatic Dis. 2013, 16, 7−15. (16) Siegel, R.; Ma, J.; Zou, Z.; Jemal, A. Cancer statistics, 2014. CaCancer J. Clin. 2014, 64, 9−29. (17) Boas, U.; Christensen, J. B.; Heegaard, P. M. H. Dendrimers: Design, Synthesis and Chemical Properties. In Dendrimers in medicine and biotechnology: New molecular tools; Boas, U., Christensen, J. B., Heegaard, P. M. H., Eds.; RSC: 2006; pp 1−27. (18) Liu, J.; Gray, W. D.; Davis, M. E.; Luo, Y. Peptide- and saccharide-conjugated dendrimers for targeted drug delivery: A concise review. Interface Focus 2012, 2, 307−24. (19) Gu, Z. W.; Luo, K.; She, W. C.; Wu, Y.; He, B. New-generation biomedical materials: Peptide dendrimers and their application in biomedicine. Sci. China: Chem. 2010, 53, 458−478. (20) Bi, X.; Shi, X.; Baker, J. R., Jr. Synthesis, characterization and stability of a luteinizing hormone-releasing hormone (lhrh)-functionalized poly(amidoamine) dendrimer conjugate. J. Biomater. Sci., Polym. Ed. 2008, 19, 131−142. (21) Boyd, B. J.; Kaminskas, L. M.; Karellas, P.; Krippner, G.; Lessene, R.; Porter, C. J. H. Cationic poly-l-lysine dendrimers: Pharmacokinetics, biodistribution, and evidence for metabolism and bioresorption after intravenous administration to rats. Mol. Pharmaceutics 2006, 3, 614−627. (22) Toth, I. A novel chemical approach to drug delivery: Lipidic amino acid conjugates. J. Drug Target. 1994, 2, 217−239.

AUTHOR INFORMATION

Corresponding Author

*Istvan Toth. E-mail: [email protected]. Phone: +61 7 3346 9892. ORCID

Istvan Toth: 0000-0002-4572-397X Present Addresses ¶

P.V.: Faculty of Pharmacy, The University of Sydney, Sydney, Australia. # A.R.: University of Waterloo, School of Pharmacy, Kitchener, Canada. ∇ F.M.M.: ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Children’s Cancer Institute, Lowy Cancer Research Centre and Australian Centre for NanoMedicine, UNSW, Sydney, Australia. Author Contributions ∥

P.V. and A.R. are co-first authors: A.R. co-designed and synthesized the compounds, and P.V. designed and performed the biological experiments. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ADT, androgen deprivation therapy; ANOVA, analysis of variance; AUC, area under the curve; CSS, charcoal stripped serum; CRPC, castration-resistant prostate cancer; Dde, 4,4dimethyl-2,6-dioxocyclohex-1-ylidine)ethyl; DHT, dihydrotestosterone; DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; E2, 17β-estradiol; ESI-MS, electrospray ionization mass spectrometry; FBS, fetal bovine serum; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; HBSSHEPES, Hank’s balanced salt solution−4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; HBTU, N,N,N′,N′-tetramethylO-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HPLC, high performance liquid chromatography; HR-MS, high resolution mass spectrometry; LC-MS, liquid chromatography−mass spectrometry; LH, luteinizing hormone; MBHA, 4-methylbenzhydrylamine; MTT, (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide); Papp, apparent permeability; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; RP-HPLC, reverse-phase high performance liquid chromatography; TR, retention time; RT, room temperature; SPPS, solid-phase peptide synthesis; t1/2, half-life; Trt-Mpa, S-trityl-3-mercaptopropionic acid

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REFERENCES

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Development of New Gonadotropin-Releasing Hormone-Modified

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