LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo.

Small RNA molecules such as microRNA and small interfering RNA (siRNA) have become promising therapeutic agents because of their specificity and their potential to modulate gene expression. Any gene of interest can be potentially up- or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability, and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the low-density lipoprotein receptor (LDLR), and their biological evaluation both in vitro and in vivo.

LDL receptor-peptide conjugate as in vivo tool for specific targeting of pancreatic ductal adenocarcinoma.

Despite clinical advances in diagnosis and treatment, pancreatic ductal adenocarcinoma (PDAC) remains the third leading cause of cancer death, and is still associated with poor prognosis and dismal survival rates. Identifying novel PDAC-targeted tools to tackle these unmet clinical needs is thus an urgent requirement. Here we use a peptide conjugate that specifically targets PDAC through low-density lipoprotein receptor (LDLR). We demonstrate by using near-infrared fluorescence imaging the potential of this conjugate to specifically detect and discriminate primary PDAC from healthy organs including pancreas and from benign mass-forming chronic pancreatitis, as well as detect metastatic pancreatic cancer cells in healthy liver. This work paves the way towards clinical applications in which safe LDLR-targeting peptide conjugate promotes tumor-specific delivery of imaging and/or therapeutic agents, thereby leading to substantial improvements of the PDAC patient's outcome.

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals (ldlr-/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

The antidepressant-like effects of the 3ß-hydroxysteroid dehydrogenase inhibitor trilostane in mice is related to changes in neuroactive steroid and monoamine levels.

In the present study, we analyzed the effects of a systemic treatment with the competitive 3ß-hydroxysteroid dehydrogenase (3ß-HSD) inhibitor trilostane on: (i) neurosteroid and monoamine levels in the brain, and (ii) the antidepressant activity of steroids and antidepressants in the forced swimming test (FST). 3ß-HSD converts pregnenolone (PREG) into progesterone (PROG) or dehydroepiandrosterone (DHEA) into androstenedione. These neuroactive steroids are known to regulate neurotransmitters effects in the brain, particularly glutamate, γ-aminobutyric acid (GABA) and serotonin (5-HT), with consequences on mood and depression. We previously reported that trilostane showed antidepressant-like properties in the FST and concomitantly regulated plasma adrenocorticotropin (ACTH) and corticosterone levels, markers of the stress-induced hypothalamus-pituitary-adrenal (HPA) axis activation. We here observed that adrenalectomy/castration blocked the trilostane effect, outlining the importance of peripheral steroid levels. Trilostane (25 mg/kg) decreased hippocampus PROG contents and paradoxically increased circulating PROG levels. It also increased PREG levels in the hippocampus and frontal cortex. In the FST, a co-treatment with trilostane facilitated DHEAS (5-20 mg/kg) antidepressant activity, but showed only an additive, not facilitative, effect with PREGS (10-40 mg/kg), PROG (10-40 mg/kg) or allopregnanolone (ALLO, 1-8 mg/kg). Trilostane (25 mg/kg) treatment significantly increased 5-HT and (-)-norepinephrine (NE) turnovers in the hippocampus, an effect likely related to its antidepressant action. In co-administration studies, trilostane further decreased immobility following fluoxetine (30-60 mg/kg), sertraline (20-40 mg/kg) and imipramine (20-40 mg/kg), but not desipramine (20-40 mg/kg), treatments. A significant additive effect was observed for the selective 5-HT reuptake inhibitors (SSRI) at their highest dose. This study confirmed that a systemic administration of trilostane directly affected peripheral and brain levels in neuroactive steroids and monoamine turnover, resulting in antidepressant activity. The drug could be proposed as a co-treatment with SSRI. This article is part of a Special Issue entitled 'Anxiety and Depression'.

The antidepressant-like effect of the 3ß-hydroxysteroid dehydrogenase inhibitor trilostane involves a regulation of ß-type estrogen receptors.

RATIONALE: Trilostane is a competitive inhibitor of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), which notably converts pregnenolone into progesterone or dehydroepiandrosterone into androstenedione. Trilostane shows antidepressant-like properties in the forced swimming test (FST). The compound, however, induced only moderate effects on neuroactive steroid levels that could be related to its behavioral efficacy. METHODS: We compared the behavioral effect of trilostane with the other 3ß-HSD inhibitor, cyanoketone, and analyzed the putative involvement of the ß-type estrogen receptor (ERß) in its antidepressant effect. RESULTS: Trilostane reduced immobility in the FST significantly at 12.5 and 25 mg/kg subcutaneously (s.c.), whereas cyanoketone (0-100 mg/kg s.c.) was ineffective. The negative ER modulator fulvestrant (ICI 182780) dose-dependently blocked the effect of trilostane (25 mg/kg). Trilostane increased circulating estradiol levels in the 12.5-50 mg/kg dose-range, and this effect was unaffected by stress and not shared by cyanoketone (25 mg/kg). The trilostane (25 mg/kg) treatment increased the ERß mRNA expression in adrenals (+100%) and centrally, in the hippocampus (+330%). Stress and cyanoketone failed to affect ERß mRNA levels in periphery or in the brain. CONCLUSIONS: These data demonstrate that the antidepressant-like potential of trilostane is not due to its 3ß-HSD inhibiting activity, since it is not shared by cyanoketone, but rather to its estrogenic activity. The compound, which releases estradiol and up-regulates ERß receptors, could be used as a therapeutic tool to allow an estrogenic facilitation of antidepressant efficacy.

The 3beta-hydroxysteroid dehydrogenase inhibitor trilostane shows antidepressant properties in mice.

Changes in neuro(active)steroid levels are involved in depressive states and mood disorders. For instance, dehydroepiandrosterone or pregnenolone sulfate showed anti-stress and antidepressant activity in rodents and regulation of allopregnanolone levels appeared to be one of the consequence of an effective antidepressant therapy in patients. 4alpha,5-Epoxy-17beta-hydroxy-3-oxo-5alpha-androstane-2alpha-carbonitrile (trilostane) inhibits the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) that, in particular, converts pregnenolone into progesterone. We examined whether systemic administration of trilostane affects the response to stress and depression. An acute treatment with trilostane (6.3-50mg/kg SC injected twice -16 and -2h before the measure) increased 3beta-HSD mRNA levels in the hippocampus and adrenals, but had little effect on protein levels. The trilostane treatment failed to affect open-field, locomotor or exploratory behaviors, but significantly reduced the immobility duration in the forced swimming test, measuring antidepressant-like activity, and increased the time spent in open arm in the elevated plus-maze, measuring anxiety response. The antidepressant-like effect of trilostane was effective after a repeated treatment (2.5-20mg/kgSC twice-a-day during 7 days) or in mice submitted to a restraint stress during 21 days and showing several behavioral and physiological parameters of depression (decreased body weight, increased adrenal glands weight and anhaedonia). Trilostane also reduced stress-induced increase in plasma corticosterone and ACTH levels, showing direct effect on HPA axis activity. These observations suggest that the 3beta-HSD inhibitor trilostane present antidepressant-like activity, putatively by regulating brain and peripheral levels of neuroactive steroids.

A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters.

Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R=0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.

Improved brain uptake and pharmacological activity profile of morphine-6-glucuronide using a peptide vector-mediated strategy.

Morphine-6-glucuronide (M6G), an active metabolite of morphine, has been shown to have significantly attenuated brain penetration relative to that of morphine. Recently, we have demonstrated that conjugation of various drugs to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated morphine-6-glucuronide to a peptide vector SynB3 to enhance its brain uptake and its analgesic potency after systemic administration. We show by in situ brain perfusion that vectorization of M6G (Syn1001) markedly enhances the brain uptake of M6G. This enhancement results in a significant improvement in the pharmacological activity of M6G in several models of nociception. Syn1001 was about 4 times more potent than free M6G (ED(50) of 1.87 versus 8.74 micromol/kg). Syn1001 showed also a prolonged duration of action compared with free M6G (300 and 120 min, respectively). Furthermore, the conjugation of M6G results in a lowered respiratory depression, as measured in a rat model. Taken together, these data strongly support the utility of peptide-mediated strategies for improving the efficacy of drugs such as M6G for the treatment of pain.

The use of cell-penetrating peptides for drug delivery.

In the past decade, several peptides that can translocate cell membranes have been identified. Some of these peptides, which can be divided into different families, have short amino acid sequences (10-27 residues in length) and enter the cell by a receptor-independent mechanism. Furthermore, these peptides are capable of internalizing hydrophilic cargoes. Although the detailed mechanism by which these molecules enter cells is poorly understood, their ability to traverse the membrane into the cytoplasm has provided a new and powerful biological tool for transporting drugs across cell membranes.

Peptide-vector strategy bypasses P-glycoprotein efflux, and enhances brain transport and solubility of paclitaxel.

We present the results obtained with paclitaxel coupled to a peptide-vector SynB3 (PAX-OSUC-SynB3), showing that this peptide-vector enhances the solubility of paclitaxel and its brain uptake in mice using the in situ brain perfusion model. We also show by the in situ brain perfusion in P-glycoprotein (P-gp)-deficient and wild-type mice that vectorized paclitaxel bypasses the P-gp present at the luminal side of the blood-brain barrier. The effect of the vectorized paclitaxel on various cancer cells was not significantly different from that of free paclitaxel. These results indicate that vectorization of paclitaxel may have significant potential for the treatment of brain tumors.

2-Pyrrolinodoxorubicin and its peptide-vectorized form bypass multidrug resistance.

A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein, which is capable of lowering intracellular drug concentrations. In the present study, we tested the capability of 2-pyrrolinodoxorubicin (p-DOX), a highly potent derivative of DOX, to bypass multidrug resistance. The accumulation, intracellular distribution and cytotoxicity of p-DOX were tested in two cell lines (K562 and A2780) and their DOX-resistant counterparts (K562/ADR and A2780/ADR). Cellular accumulation and cytotoxicity were dramatically lowered for DOX in resistant cell lines, in comparison with non-resistant cells. In contrast, cellular accumulation, intracellular distribution and cytotoxicity of p-DOX were independent of the nature of the cell lines. The p-DOX showed potent dose-dependent inhibition of cell growth against resistant cells as compared with DOX. After treatment of resistant cells with verapamil, the intracellular levels of DOX were markedly increased and consequent cytotoxicity improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement of cell uptake or an increase in the cytotoxic effect of the derivative p-DOX, indicating that the compound bypasses the P-glycoprotein. Finally, we show that vectorization of p-DOX by a peptide vector (SynB3) which has been shown to enhance the brain uptake of DOX and to decrease its heart accumulation does not affect this property. These results indicate that p-DOX and its vectorized form are potent and effective in overcoming multidrug resistance.

Evidence for an active transport of morphine-6-beta-d-glucuronide but not P-glycoprotein-mediated at the blood-brain barrier.

Morphine-6-beta-d-glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood-brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P-glycoprotein (P-gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance-associated protein mrp1 and the glucose transporter GLUT-1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild-type and deficient mice [mdr1a(-/-) and mrp1(-/-)], with and without probenecid, digoxin, PSC833 or d-glucose. No difference was found between P-gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co-perfused with probenecid in wild-type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co-perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P-gp and sensitive to digoxin and PSC833, may be involved. The co-perfusion of [14C]M6G with d-glucose revealed a threefold decrease in M6G uptake. In conclusion, P-gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT-1 and a digoxin-sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.

Studies on the internalization mechanism of cationic cell-penetrating peptides.

A great deal of data has been amassed suggesting that cationic peptides are able to translocate into eucaryotic cells in a temperature-independent manner. Although such peptides are widely used to promote the intracellular delivery of bioactive molecules, the mechanism by which this cell-penetrating activity occurs still remains unclear. Here, we present an in vitro study of the cellular uptake of peptides, originally deriving from protegrin (the SynB peptide vectors), that have also been shown to enhance the transport of drugs across the blood-brain barrier. In parallel, we have examined the internalization process of two lipid-interacting peptides, SynB5 and pAntp-(43-58), the latter corresponding to the translocating segment of the Antennapedia homeodomain. We report a quantitative study of the time- and dose-dependence of internalization and demonstrate that these peptides accumulate inside vesicular structures. Furthermore, we have examined the role of endocytotic pathways in this process using a variety of metabolic and endocytosis inhibitors. We show that the internalization of these peptides is a temperature- and energy-dependent process and that endosomal transport is a key component of the mechanism. Altogether, our results suggest that SynB and pAntp-(43-58) peptides penetrate into cells by an adsorptive-mediated endocytosis process rather than temperature-independent translocation.

Improved brain uptake and pharmacological activity of dalargin using a peptide-vector-mediated strategy.

The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.

Induction of antigen-specific CTL responses using antigens conjugated to short peptide vectors.

Linear peptides (SynB vectors) with specific sequence motifs have been identified that are capable of enhancing the transport of a wide range of molecules into cells. These peptide vectors have been used to deliver exogenous peptides and protein Ags across the cell membrane and into the cytoplasm of cells. Specifically, in vitro analysis indicated that these SynB peptides enhanced the uptake of two 9-mer peptide Ags, NP(147-155) and Mtb(250-258) (T cell epitopes of influenza nucleoprotein and Mycobacterium tuberculosis, respectively) and the M. tuberculosis Ag Mtb8.4 protein, into K562 cells when covalently linked to the respective Ags. Furthermore, selected SynB vectors, when conjugated to these same Ags and used as immunogens, resulted in considerably enhanced Ag-specific CTL responses. Several SynB vectors were tested and resulted in varying levels of cellular uptake. The efficiency of uptake correlated with the ability of the SynB construct to deliver each epitope in vivo and induce specific CTL responses in mice. These data suggest that peptide vectors, such as SynB that transport target Ags across the cell membrane in a highly efficient manner, have significant potential for vaccine delivery.

Improved brain delivery of benzylpenicillin with a peptide-vector-mediated strategy.

Previous studies from our laboratory have demonstrated that the coupling of doxorubicin with SynB1 vector dramatically increases its brain uptake. In the present study, we have evaluated the broad application of this approach using another molecule: benzylpenicillin (B-Pc). We, therefore, have coupled the beta-lactam antibiotic B-Pc with SynB1 and assessed its ability to cross the blood-brain barrier (BBB) using the in situ rat brain perfusion method. We first confirmed the very low brain uptake of free radiolabeled B-Pc. When B-Pc was coupled to SynB1, its uptake in brain was increased by a factor of 7, without compromising the BBB integrity. The vectorised B-Pc was distributed in all the gray areas assessed (frontal, parietal, and occipital cortex, thalamus, hippocampus, and striatum). Moreover, using a wash-out procedure and a capillary depletion method, we have shown that the radiolabeled B-Pc was associated mainly with brain parenchyma. In summary, this study demonstrates the successful application of the use of SynB1 vector for the transport of B-Pc across the BBB.

Translocation of protegrin I through phospholipid membranes: role of peptide folding.

The protegrin PG-1, belonging to the family of beta-stranded antimicrobial peptides, exerts its activity by forming pores in the target biological membranes. Linear analogues derived from PG-1 do not form pores in the phospholipid membranes and have been used successfully to deliver therapeutic compounds into eucaryotic cells. In this paper, the translocation of PG-1 and of a linear analogue through artificial phospholipid membranes was investigated, leading to a possible mechanism for the activity of these peptidic vectors. We report here that [12W]PG-1, a fluorescent analogue of PG-1, is able to translocate through lipid bilayers and we demonstrate that this property depends on its secondary structure. Our results agree with the recent mechanism proposed for the translocation and permeabilisation activities of several helical and beta-stranded peptides. In addition, our data corroborate recent work suggesting that certain protegrin-derived vectors enter into endothelial cells by adsorptive-mediated endocytosis.

Peptide delivery to the brain via adsorptive-mediated endocytosis: advances with SynB vectors.

Biological membranes normally restrict the passage of hydrophilic molecules. This impairs the use of a wide variety of drugs for biomedical applications. To overcome this problem, researchers have developed strategies that involve conjugating the molecule of interest to one of a number of peptide entities that are efficiently transported across the cell membranes. In the past decade, a number of different peptide families with the ability to cross the cell membranes have been identified. Certain of these families enter the cells by a receptor-independent mechanism, are short (10-27 amino acid residues), and can deliver successfully various cargoes across the cell membrane into the cytoplasm or nucleus. Surprisingly, some of these vectors, the SynB vectors, have also shown the ability to deliver hydrophilic molecules across the blood-brain barrier, one of the major obstacles to the development of drugs to combat diseases affecting the CNS.