Field of the invention

The invention relates to a CPP, preferably a cationic CPP for use as a medicament for treating cancer or a pre-malignant disorder wherein said CPP is able to induce the formation of a ceramide.

Background of the invention

Interest in cell-penetrating peptides (CPPs) as tools to deliver membrane-impermeable therapeutic molecules continues to increase. This development is illustrated by the expanding efforts of the scientific community to elucidate the chemical, physical and biological characteristics of many CPPs and CPP-based delivery systems (Magzoub and Graslund, 2004; Morris et al, 2008; Patel et al, 2007; Fischer et al, 2005a) and the steep rise in the number of therapeutic strategies that are being pursued with these systems (Foerg and Merkle, 2008; Stewart et al, 2008). It is becoming progressively clear that instead of a single mechanism that is valid for all CPPs, multiple modes of actions exist with respect to the route of internalization and the intracellular trafficking that depend on the cell line (Mueller et al, 2008), the CPP (Duchardt et al, 2007) and the cargo (Lundin et al, 2008; Tunnemann et al, 2006). Although endocytosis is now considered the major internalization route for cationic CPPs, (Kaplan et al, 2005; Nakase et al, 2004; Richard et al, 2005; Wadia et al, 2004; Fischer et al, 2005a) direct translocation for cationic CPPs has also been observed, both at high and at low concentrations.

The potential for direct translocation is thought be related to the ability of the guanidinium moieties of arginines to form bidentate hydrogen bonds with membrane lipids and is underscored by the ability of the arginine-rich TAT peptide to induce pores in artificial membranes, as was observed by small angle X-ray scattering (Mishra et al., 2008). Moreover, molecular-dynamics simulations indicate the potential of arginine-rich peptides to form transient pores in plasma membranes in the presence of an electrochemical gradient and provide a mechanistic hypothesis for direct membrane translocation. Nevertheless, biological details remain poorly understood. At higher concentrations, direct cytoplasmic import of cationic CPPs has been shown to depend on spatially confined nucleation zones, which are sensitive to the PKC5 inhibitor rottlerin and to chlorpromazine. Currently, CPPs have been exclusively applied for their main pharmacokinetic property - their ability to improve cargo-delivery.

Relatively little is known regarding their pharmacodynamic properties. In the developed world, one in three people will develop cancer during their life. Several treatments have already been developed. Some of them induce unwanted side effects such as toxicity. There is still a need for new cancer treatments.

Description of the invention

We discovered that a cationic CPP is able to induce the formation of a ceramide. This finding opens the possibility to use cationic CPPs for treating cancer. The inventors demonstrated that cationic CPPs induced the activity of acid sphingomyelinase (ASMase) at the plasma membrane and that this is coupled to their uptake. The rationale for investigating a possible role of ASMase in peptide uptake was that two known inhibitors of ASMase activity - rottlerin and chlorpromazine - inhibited peptide uptake via direct translocation at high peptide concentrations.

Acid sphingomyelinase has traditionally been known in relation to Niemann-Pick disease, a lysosomal storage disorder. More recently, an important role for this enzyme in ceramide-mediated signal transduction pathways has emerged, linking its activity to a variety of common diseases, among which are cancer, cardiovascular diseases and diabetes. A major function of this enzyme is the hydrolysis of sphingomyelin to ceramide and phosphorylcholine following translocation from lysosomes to the outer leaflet of the plasma membrane.

Ceramide is a lipid with a well-established role in the induction of antiproliferative and apoptotic responses in a variety of cancer cells. As a part of this role, ceramide greatly affects the structure and properties of cellular membranes. In the plasma membrane ceramide-enriched membrane domains modulate signaling. In the mitochondrial outer membrane ceramide increases permeability (Siskind et al., 2006). Here, we provide evidence that ceramide formation by ASMase at the plasma membrane plays an important role in the rapid cytoplasmic import of cationic CPPs. This finding gives rise to the concept that cationic CPPs enhance their own uptake by initiating a positive feed-back loop that involves an enzymatic alternation of the lipid composition of the plasma membrane. Accordingly, in a first aspect, there is provided a CPP, preferably a cationic CPP for use as a medicament for treating cancer or a pre-malignant disorder wherein said CPP is able to induce the formation of a ceramide.

Within the context of the invention, a ceramide is defined as being a family of lipid molecules found within the plasma membrane. A ceramide is composed of sphingosine and a fatty acid.

The formation of ceramide is preferably assessed using the diacylglycerol kinase assay (Grassme et al, 2005).

Alternatively, the induction of a ceramide formation may also be indirectly assessed by assessing the activity of an ASMase. An induction of an ASMase activity is expected to induce a ceramide. An alteration of an ASMase activity may be assessed by an change of its cellular localization: ASMase is translocated from an intra-cellular compartment where it is inactive to the outer leaflet of the plasma membrane following its activation (Zeidan and Hannun, 2007), Subsequently this enzyme changes the plasma membrane lipid composition by hydrolyzing sphingomyelin to ceramide. The assessment of the cellular localization of an ASMase may be carried out using immunofluoresence, preferably as described in the experimental part. Alternatively, the activity of ASMase can be assessed using a commercial acid sphingomyelinase assay kit or by following the conversion of [ ¹⁴ C] in the assay as described by Grassme et al. (Grassme et al, 2005).

A CPP, preferably a cationic CPP is preferably said to induce the formation of a ceramide when a significant increase of ceramide is detected using the assay as defined earlier herein by comparison to a control wherein no CPP, preferably no cationic CPP has been added. A detectable increase is preferably an increase of at least 10%, 20%, 30% 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Alternatively a CPP, preferably a cationic CPP is preferably said to induce the formation of a ceramide when a significant increase of an ASMase activity is detected in the assay as defined earlier herein by comparison to a control wherein no CPP, preferably no cationic CPP has been added. A detectable increase is preferably an increase of at least 50%> 55%, 60%, 65%, 70% or 75%, or more. A CPP may be any CPP known to the skilled person. A CPP is preferably a cationic CPP. Any cationic CPP known to the skilled person may be used in the present invention. A CPP may be defined as a peptide of a given size, amino acid sequence and charge which stimulates its own uptake by activating one or more endocytosis routes and/or directly translocates through the plasma membrane and has therefore the potential to enhance the delivery of a molecule or nanometer-sized delivery construct into the cell, to the cytoplasm or an organelle (Fischer et al., 2005b) so that uptake occurs to a greater degree than would be expected if the CPP was not present. A preferred way, but not the only way, of assessing whether a peptide can operate as a CPP is through complexation - either covalent or non-covalent - with a fluorescently labeled macromolecule and to assess whether the complexation enhances the cellular internalization of the fluorescently labeled macromolecule (Duchardt et al, 2009a). A preferred size is from 6-60 amino acids or from 6 to 30 or from 6 to 25 or from 8 to 22 amino acids.

A CPP preferably has an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or preferably arginine. In this context, "high" preferably means that a CPP has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of positively charged amino acids such as lysine and/or arginine. In a preferred embodiment, a CPP has a size of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 amino acids and comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 positively charged amino acids. More preferably, a CPP has a size of 22 amino acids and has 7 positively charged amino acids such as 4 arginine and 3 lysine residues.

Alternatively, a CPP has a sequence that contains a pattern of polar/charged amino acids such as arginine and/or lysine and non-polar, hydrophobic amino acids such as leucine, isoleucine, phenylalanine and/or valine that is considered amphipathic by those familiar with the art.

These two types of structures are referred to as polycationic or amphipathic, respectively by the skilled person. However, in the context of the invention we preferably name so-called polycationic or amphipathic CPP a cationic CPP.

It is understood for a skilled person, that a cationic CPP may comprise or consist totally or in part out of L-amino acids and/or D-amino acids. Furthermore, the sequence of D- amino acids may also be in reverse order. To the skilled person, such a peptide is referred to as a retro-inverso analog. A retro-inverso analog (D-amino acids) has the same orientation of the side chains relative to the other side chains as the normal L- peptide and as such has a larger topographical equivalence to the normal L-peptide than a D-peptide of the same sequence. The relative orientation of the side chains to each other is changed significantly if the sequence is changed from L- to D-amino acids without inverting the sequence. This is true because L- and D-amino acids are enantiomers (analogy with left and right hand). The use of such retro-inverso analog is quite attractive since stability is expected to be strongly improved. L-peptides are degraded by proteases very rapidly, whereas D-peptides cannot be degraded by proteases at all (or only very slowly). Of course, the improvement of the stability might also positively affect the 'final' efficacy. Specificity is expected to be the same.

Preferably, a cationic CPP is selected from the group consisting of: a cationic CPP peptidomimetic, an arginine-rich CPP, a guanidine-rich CPP peptidomimetic, a CPP derived from the human milk protein lactoferrin. A preferred amino acid sequence of the human milk protein lactoferrin is: KCFQWQR MRKVRGPPVSCIK (SEQ ID NO: l) .

A cationic CPP peptidomimetic is a 6-60-mer peptide-like oligomer designed to mimic a CPP. Other sizes are also encompassed by the invention as earlier defined herein. These molecules may be derived from an existing CPP by introducing a backbone modification in order to alter the molecule's properties. For example, they may correspond to modifications to change the molecule's stability or biological activity. These modifications involve changes to the peptide that will not occur naturally (such as altered backbones). The introduction of a six-methylene spacer increased uptake efficiency (Wender et al, 2000), whereas the introduction of a 6-aminohexanoic acid between arginines in the backbone increased the cytosolic delivery of steric block morpholine oligomers (Abes et al, 2008).

An arginine-rich CPP preferably comprises or contains or consists of at least 20 % and preferably more than 50%, 60%, 70%, 80%, 90%, 100% of arginine residues or preferably at least 3, 4, 5, 6, 7, 8 arginine residues. Preferably such arginine- rich CPP comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 amino acids. Examples of arginine-rich CPP include, but are not limited to, a peptide comprising or consisiting of tat 49-57 (SEQ ID NO:2: RKKRRQRRR), nona-arginine (SEQ ID NO:3: RRRRRRRRR or R9) and penetratin (SEQ ID NO:4: RQIKIWFQNRRMKWKK). Good results were obtained with R9 consisting of L-amino acids identified as R9 or L- R9. Good results were also obtained with R9 consisting of D-amino acids identified as r9 or D-R9.

A guanidine-rich CPP peptidomimetic preferably comprises or contains or consists of at least 4 guanidino-groups. A CPP derived from a human milk protein lactoferrin is another preferred embodiment of the invention. A human lactoferrin protein is represented by SEQ ID NO: 1.

Preferably said CPP is as identified in WO 2007/076904. Preferred CPP comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 contiguous amino acids from a human lactoferrin and/or at least four, 5, 6, 7, 8 cationic amino acids or more depending on the total number of amino acids of the CPP. In this embodiment, it is preferred that a CPP derived from a human milk protein lactoferrin is not a human lactoferrin protein, more preferably said CPP is not the human milk protein lactoferrin represented by SEQ ID NO: 1.

In a preferred embodiment, a cationic CPP is not the pl4ARF as disclosed in WO 2008/063113. These specific CPP are able to induce apoptosis by mimicking the function of the tumor suppressor protein pi 4ARF, which is a mechanism unrelated to the formation of ceramide.

Our invention is preferably not using a CPP consisting of 15 to 25 amino acids comprising at least amino acids 1 - 14 of a mature mammalian tumor suppressor protein ARF or a partially inverted sequence thereof. Preferably, our invention is not using a sequence derived from a human pl4ARF protein (such as SEQ ID NO:5), or a mouse pl9ARF protein (such as SEQ ID NO: 6) or a partially inverted sequence thereof. Preferably, our invention is not using the following peptide sequence derived from a human pl4ARF protein (such as SEQ ID NO: 7).

The use of a CPP, preferably a cationic CPP as identified herein preferably leads to a therapeutic effect. Ceramide, as alluded to before, is downregulated in many tumors due to the hypersensitivity of tumours to ceramide. The negative membrane potential of cancer cell membranes, together with said hypersensivity, is expected to result in a broad applicability of a CPP, preferably a cationic CPP as an inducer of ceramide in cancer cells. A therapeutic effect may be an anti-tumor effect. An anti-tumor effect is preferably identified as:

- an inhibition or proliferation of tumor cells and/or

- an induction or increased induction of tumor cells death and/or

- an inhibition or prevention or delay of the increase of a tumor weight or growth and/or

- a prolongation of patient survival of at least one month, several months or more (compared to those not treated or treated with an isotype control).

In the context of the invention, a patient may survive and/or may be considered as being disease free. Alternatively, the disease or condition may have been stopped or delayed.

An inhibition of the proliferation of tumor cells may be at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more. Proliferation of cells may be assessed using known techniques. An induction of tumor cell death may be at least 20%, 25%, or more. Tumor growth may be inhibited at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75% or more. Tumor cell death may be assessed using techniques known to the skilled person. Tumor cell death may be assessed using MRI (Magnetic Resonance Imaging) or CT

(Computer Tomography).

In certain embodiments, tumor weight increase may be inhibited by at least 20% or more. In certain embodiments, tumor growth may be delayed by at least one week, one month, two months or more. Tumor weight or tumor growth may be assessed using techniques known to the skilled person.

The detection of tumor growth or the detection of the proliferation of tumor cells may be assessed in vivo by measuring changes in glucose utilization by positron emission tomography with the glucose analogue 2-[18F]-fluor-2-deoxy-D-glucose (FDG-PET) or [18F]-'3-fluoro-'3-deoxy-L-thymidine PET.

In each embodiment wherein the effect of a CPP, preferably a cationic CPP is quantified, the assay may be carried out by comparison to a subject not treated or to the same subject before treatment or compared to a subject treated with control compound. A tumor can be a solid tumor or a non-solid tumor such as lymphoma. Some types of tumors that can be treated using the present invention are extensively identified later herein.

The dosage for a CPP, preferably a cationic CPP of the invention can be readily determined by extrapolation from the in vitro tests and assays described below, or from animal experiments or from human clinical trials. In a preferred embodiment, a dose of at least 50 μg of a CPP, preferably a cationic CPP is administered, preferably at least 250μg, at least 1 mg, at least 5 mg or with a higher concentration as identified from a dose-escalation study.

A subject that can be treated with a CPP, preferably a cationic CPP includes, but is not limited to a subject that has been diagnosed as having a cancer, a pre-malignant disorder. Examples of cancer include brain cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colorectal cancer, colon cancer, gynecologic tumors (e. g. uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva, HPV derived cancer), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e. g. , cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, leukemia, myeloma, multiple myeloma, cancer of the urethra, cancer of the penis, prostate cancer,chronic or acute leukemia, solid tumors of childhood, Hodgkin's disease, lymphocytic lymphomas, non- Hodgkin lymphoma, cancer of the bladder, liver cancer, renal cancer, cancer of the kidney or ureter (e. g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e. g., primary CNS lymphoma, spinal axis tumors, brain stemgliomas or pituitary

adenomas), glioma or fibrosarcoma.

As used herein, the term "subject" preferably refers to a human or a non-human mammal. Preferably a subject being treated is a human.

In a preferred embodiment, a CPP, preferably a cationic CPP is used as a sole or single component or a sole or single active ingredient for treating a cancer. Alternatively, a CPP, preferably a cationic CPP as defined herein may be used in combination with another cancer therapy or treatment. It is however important to note that the invention is the first to have unravelled that a CPP, preferably a cationic CPP can be used as an active ingredient, preferably as sole active ingredient for treating cancer via their induction of the formation of a ceramide. In an embodiment, a CPP, preferably a cationic CPP is used (simultaneously or sequentially) with another molecule and/or another treatment or therapy. Examples of another treatment or therapy include another classical cancer treatment such as chemotherapy, radiotherapy. Examples of another molecule include a DNA replication inhibitor such as cisplatin, taxol and/or another compound. A molecule used as a chemotherapeutic is preferably not linked to a CPP. Non-classical treatments may also be used. For example, non-classical treatments aimed specifically at intervening in the ceramide metabolism These include the inhibition of sphingosine-1 -kinase (Takabe et al., 2008), acid ceramidase (Zeidan et al., 2008), neutral ceramidase or glucosyl transferase (Dumitru et al, 2009), via siRNA or small-molecule strategies. Several of said strategies are presently in clinical

development (Guillermet-Guibert et al., 2009). A CPP, preferably a cationic CPP may be linked to another molecule. Further treatments for use in combination with a CPP include, but not limited to, an inhibitor of angiogenesis, an antibody and/or a kinase inhibitor as known to the skilled person. When a CPP, preferably a cationic CPP is sequentially or simultaneously used with another molecule or treatment, it may be expected that a synergistic effect is obtained. It preferably means that a given effect is obtained with a lower dose or concentration of a CPP, preferably a cationic CPP and/or of the other molecule or treatment compared to the same effect obtained with a higher concentration or dose of a same CPP, preferably a cationic CPP used alone or with a higher concentration or dose of the other molecule or treatment alone. More preferably another molecule in this context is a chemotherapeutic. Even more preferably, a same effect is obtained using a dose or concentration of said chemotherapeutic which is 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50% lower than a corresponding dose or concentration of said chemotherapeutic used alone without a CPP, preferably without a cationic CPP.

In a preferred embodiment a CPP, preferably a cationic CPP is used in combination with another compound or molecule, which is able to stimulate the immune system, i.e. an immune stimulatory compound, hereafter named second stimulating compound. A CPP, preferably a cationic CPP and a second stimulating compound may be administered simultaneously or sequentially. More preferably, a CPP, preferably a cationic CPP and a second stimulating compound are formulated in one single composition.

In a preferred embodiment, a CPP as defined herein is used as a sole or single component or sole or single active ingredient or is used sequentially or simultaneously with another component or active ingredient which is not linked to said CPP. Most preferably, said CPP comprises or consists of L-R9 or D-R-9. Said other component is preferably a chemotherapeutic. A CPP, preferably a cationic CPP and optionally another compound are formulated as a composition. Preferably, a composition is a pharmaceutical composition. Such a pharmaceutical composition preferably further comprises a pharmaceutical excipient and/or an immune modulator. Any known inert pharmaceutically acceptable carrier and/or excipient may be added to the composition. Formulation of medicaments, and the use of pharmaceutically acceptable excipients are known and customary in the art and for instance described in Remington; The Science and Practice of Pharmacy, 21 ^nd Edition 2005, University of Sciences in Philadelphia.

A CPP, preferably a cationic CPP and optionally another compound or component or ingredient as used in the invention are preferably soluble in physiologically acceptable watery solutions (e.g. PBS) comprising no more than 35, 20, 10, 5 or 0% DMSO.

The way of administration of a CPP, preferably a cationic CPP and optionally another compound is not critical for the invention. Ways of administration are known and customary in the art are for instance described in Remington; The Science and Practice of Pharmacy, 21 ^st Edition 2005, University of Sciences in Philadelphia. The

administration may be intravenous or subcutaneous, or intramuscular administration, although other administration routes can be envisaged, such as mucosal administration or intradermal and/or intracutaneous administration, e.g. by injection. Depending of the administration envisaged, the skilled person will choose the most suited formulation for a composition comprising a cationic CPP as identified herein.

In a further aspect, there is provided a use of a CPP, preferably a cationic CPP for the manufacture of a medicament for treating cancer or a pre-malignant disorder wherein said CPP is able to induce the formation of ceramide. Each feature of this use has already been defined herein.

Accordingly, in a further aspect, there is provided a method for treating cancer or a pre-malignant disorder, wherein a CPP, preferably a cationic CPP is used, wherein said CPP is able to induce the formation of a ceramide. Each feature of this method has already been defined herein. In an embodiment, said method is carried out in a cell in vitro in a sample from a subject. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a CPP as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word "approximately" or "about" when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the figures

Figure 1. Uptake of cationic cell-penetrating peptides is sensitive to imipramine and rottlerin. (A) Effect of a pre-treatment with imipramine or rottlerin on the uptake of R9 in HeLa cells. HeLa cells were pre-treated for 30 min with imipramine or rottlerin and incubated for 20 min at 37°C with R9 in the presence of the inhibitors, washed and analyzed by confocal microscopy. 0.1 % DMSO was included as a vehicle control. The scale bar corresponds to 20 μηι. (B) Quantification of cellular fluorescence by flow cytometry. Cells were incubated with 20 or 10 μΜ R9 for 20 min at 37°C with (no fill) or without (grey) imipramine pre-treatment. (C) Median fluorescence intensity of uptake of R9 with (+) or without (-) imipramine as derived from flow cytometry. Errors bars indicate standard error of the mean. (D) Effect of imipramine on the uptake of the cationic CPP hLF at 5 and 20 μΜ. Experimental conditions were similar as described above. The error bar indicates 20 μιη.

Figure 2. Reduced uptake of R9 in imipramine-treated HeLa cells can be rescued in a dose-dependent manner by external sphingomyelinase. HeLa cells were seeded in 8- well microscopy chambers and grown to 75 % confiuency. Where indicated, cells were pre-treated with 30 μΜ imipramine for 30 min and the effect of addition of various concentrations of bacterial sphingomyelinase (bSMase) on uptake of 20 μΜ R9 was evaluated. Cells were incubated with peptide and bSMase for 20 min at 37°C and immediately analyzed by confocal microscopy. The scale bar corresponds to 40 μιη.

Figure 3. R9 induces the translocation of ASMase to and the formation of ceramide- enriched membrane platforms at the extracellular leaflet of the plasma membrane. HeLa cells were seeded in 8-well microscopy chambers and grown to 75 % confiuency. Cells were treated with 20 μΜ R9 for 20 min at 37°C or left untreated. Immunofluorescence without permeabilization was performed using anti-ASMase (IgG) and anti-ceramide (IgM) antibodies, including isotype controls. Alexa-633 conjugated secondary antibodies were visualized by confocal microscopy. The scale bar corresponds to 20 μιη.

Figure 4. Bacterial sphingomyelinase (bSMase) lowers the threshold of uptake via nucleation zones for R9 and Smac-R9, but not for R9-HPMA. HeLa cells were seeded in 8-well microscopy chambers and grown to 75 % confiuency. Cells were incubated for 30 min with the indicated concentrations of R9 or R9-conjugates +/- 1000 mU bSMase, washed and analyzed by confocal microscopy. The scale bar corresponds to 20 μιη. Figure 5. Schematic overview depicting the proposed import mechanism of cationic CPPs via direct translocation. (A) First, CPPs induce the translocation of ASMase from intracellular compartments to the outer leaflet of the plasma membrane via an unknown mechanism. (B) Second, ASMase hydrolyzes sphingomyelin to phosphorylcholine and ceramide, leading to ceramide-enriched microdomains that may fuse to form larger ceramide-enriched membrane platforms (C) Third, cationic CPPs enter the cytosol directly via the plasma membrane, most likely at the border region of segregated gellike ceramide rich domains and more fluid domains, as this border region is expected to be the most permeable part of the plasma membraneln their combination, these steps constitute a positive feed-back loop.

Figure 6. In vitro cell cytotoxicity assay showing enhanced sensitivity to

chemotherapeutics of cancer cells in the presence of D-R9 or L-R9 . Cell cytotoxicity was measured with Resazurin Assay . A. Ovcar-3 cells were treated with indicated Taxol concentrations, alone (□ ) or together with D-R9 (111 - 2μΜ,■ - 6 μΜ) for 24 hours. Data represents average of 3 independent experiments carried out in triplicate. Error bars represent mean ± SEM. Significant differences between Taxol and Taxol + D-R9 are indicated by a star (p<0.05) B. Ovcar-3 cells were treated with indicated Taxol concentrations, alone (□ ) or together with L-R9 (111 - 2μΜ,■ - 6 μΜ) for 24 hours. Data are representative of 2 independent experiments carried out in triplicate. Error bars represent ± SEM. Significant differences between Taxol and Taxol + L-R9 are indicated by a star ^ (p<0.05). C. HeLa cells were treated with indicated

Doxorubicin concentrations, alone (□ ) or together with D-R9 (111 - 2μΜ,■ - 6 μΜ) Cell cytotoxicity was measured with Resazurin Assay. Data represents 4 independent experiments carried out in triplicate. Error bars represent ± SEM. Significant differences between Doxorubicin and Doxorubicin + D-R9 are indicated by a star (p<0.05). Examples

Experimental procedures

Materials- C-terminally amidated peptides were purchased from EMC microcollections (Tubingen, Germany). Fluorophore-labeled peptides were synthesized with an N- terminal fluorescein-label. Purity was evaluated by high-performance liquid chromatography (HPLC) and identity confirmed by mass spectrometry. If required, peptides were further purified to a purity of > 95 % using reversed-phase HPLC. The CPP hLF was oxidized before use as described previously (Duchardt et al, 2009a). Bovine serum albumin (BSA), glucose, imipramine, rottlerin were from Sigma- Aldrich (Zwijndrecht, the Netherlands). Standard chemicals were from Sigma-Aldrich and Merck (Darmstadt, Germany).

Cell culture- HeLa cells were maintained in RPMI 1640 (PAN Biotech, Aidenbach, Germany) supplemented with 10 % fetal calf serum (PAN Biotech) and incubated at 37°C in a 5 % C0 ₂ -containing, humidified incubator. Cells were passaged every 2 to 3 days.

Confocal laser scanning microscopy- Confocal laser scanning microscopy was performed on a TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an HCX PL APO 63 x N.A. 1.2 water immersion lens. Cells were maintained at 37°C on a temperature-controlled microscope stage.

Peptide uptake - For confocal microscopy, MC57 cells, HeLa cells or ASMase- deficient orbit fibroblasts cells were seeded in 8-well microscopy chambers (Nunc, Wiesbaden, Germany) and grown to 75 % confluency. Cells were incubated with peptides in RPMI 1640 supplemented with 10 % fetal calf serum for times and concentrations as indicated for the individual experiments. Imipramine pre-treatments to reduce ASMase activity were conducted by pre-incubating cells for 30 min with 30 μΜ imipramine in RPMI 1640 without serum. 30 μΜ imipramine and 10 % serum were included during peptide incubations. Cells were washed twice after the incubation and living cells were analyzed immediately by confocal microscopy. Modifications for uptake experiments by flow cytometry were the use of 24-well plates (Sarstedt, Numbrecht, Germany) and a trypsinization step, which was followed by centrifugation and resuspension in 200 μΐ HEPES -buffered saline (HBS). Cellular fluorescence of 10,000 gated cells was measured using a BD FACScan flow cytometer equipped with a 488 laser (BD Biosciences, Erembodegem, Belgium) and analyzed using Summit software (Fort Collins, USA).

Immunofluorescence - HeLa cells were seeded in 8-well microscopy chambers and grown to 75 % confluency. Cells were incubated with 20 μΜ R9 for 20 min at 37°C, washed twice with pre-warmed HBS/BS A/glucose (10 mM HEPES, 135 mM NaCl, 5 mM KC1, 1 mM MgCl ₂ , 1.8 mM CaCl ₂ , pH 7.4 containing 0.1 % (w/v) BSA and 5 mM glucose) supplemented with 0.025% Tween-20 and fixed for 15 min at room temperature (RT) with 3 % paraformaldehyde in HBS. Then, cells were washed twice with HBS/BSA/glucose supplemented with 0.025 % Tween-20 and blocked for 15 min at RT with HBS/BSA/glucose supplemented with 0.025 % Tween-20. Incubation with the primary antibody took place for 1 h at RT in 150 μΐ/well. The rabbit polyclonal IgG anti-ASMase antibody scl l352 (diluted 1 :50 in HBS containing 0.1 % BSA, Santa Cruz Biotechnology, Santa Cruz, U.S.A.) was used for ASMase detection. A rabbit polyclonal IgG against NFKB was used as an isotype control (Santa Cruz Biotechnology). Ceramide was detected with a 1 :50 dilution in HBS/BSA of a monoclonal mouse anti-ceramide antibody 15B4 (Alexis biochemicals, Lausen, Switzerland). A b allotype, anti-KLH mouse IgM antibody (BD Pharmingen, San Diego, U.S.A.) was used as an isotype control for ceramide detection. After incubation with the primary antibody, cells were washed 2 x 5 min with HBS/BSA/glucose supplemented with 0.025 % Tween-20. Incubation with secondary antibodies was for 45 min, after which confocal images were taken immediately. For detection of ASMase and isotype control, an alexa-633 conjugated goat-anti-rabbit IgG (H+L) antibody (Molecular probes, Invitrogen, Eugene, U.S.A, 1 : 100 diluted in HBS containing 0.1 % BSA) was used. For detection of ceramide and isotype, an alexa-633 conjugated goat- anti mouse IgG (H+L) antibody (Molecular probes, 1 : 100 diluted in HBS containing 0.1 % BSA) was used. Results

Inhibition of direct translocation of R9 after inhibition of ASMase- Previously, we and others described the direct cytoplasmic import of cationic CPPs through localized plasma membrane regions, which we termed nucleation zones. This import had a particular pharmacological profile, being sensitive to the PKC5 inhibitor rottlerin, and chlorpromazine. In a search for a common denominator of these findings, we found that both compounds act as inhibitors of AMSase activity. We therefore hypothesized that the rapid cytoplasmic import might depend on the activation and translocation of the enzyme ASMase from an intracellular compartment to the extracellular leaflet of the plasma membrane, where it would change the plasma membrane composition by hydrolyzing sphingomyelin to ceramide. PKC5 has been associated with the activation of ASMase Chlorpromazine is a cationic amphiphilic drug that accumulates in acidic compartments in living cells. This sequestration leads to the displacement of ASMase from lipid membranes of acidic vesicles to the lumen, after which it is susceptible to proteolytic degradation by lysosomal proteases. To probe for the involvement of ASMase in the direct translocation of R9 in HeLa cells, we decided to use imipramine as a pharmacological ASMase inhibitor. Like chlorpromazine, imipramine is a cationic amphiphilic molecule that accumulates in lysosomal compartments. Its inhibitory effect on ASMase activity was experimentally verified by Grassme and colleagues . As predicted, we observed that the uptake of R9 was greatly decreased to a similar degree by imipramine as by rottlerin (Fig 1A). No major effects of both inhibitors were observed at a peptide concentration of 5 μΜ, suggesting that endocytosis was unaffected by these molecules. A quantitative analysis by flow cytometry confirmed that internalization was reduced several fold at 10 and 20 μΜ R9 (Fig. 1B-C). At 5 and 2.5 μΜ, the reduction of the median fluorescence was only 19 % and 22 %, respectively. The use of hLF, a cationic CPP derived from human lactoferrin hLF , demonstrated that imipramine-sensitivity of direct uptake was not restricted to R9, as an almost complete removal of cytoplasmic fluorescence at 20 μΜ was observed (Fig. ID). Similarly, hLF uptake via endocytosis at 5 μΜ seemed unaffected.

As our hypothesis implicated ceramide formation at the plasma membrane following ASMase translocation in CPP uptake, we reasoned that a direct translocation of cationic CPPs after an imipramine treatment may be rescued by addition of exogenous bacterial sphingomyelinase. As can be seen in figure 2, the addition of bSMase clearly rescued the cytoplasmic import of R9 in HeLa cells in a dose-dependent manner, providing more direct evidence for the notion that ceramide formation at the plasma membrane is required for the direct cytoplasmic import. Translocation of ASMase and the formation of ceramide- enriched membrane platforms- We then pursued the visualization of the translocation of ASMase to the extracellular leaflet of the plasma membrane. To achieve this, the presence of ASMase on the outer leaflet of the plasma membrane in unpermeabilized HeLa cells that were treated with R9 or left untreated was compared by immunofluorescence (Fig. 3). ASMase was detectable in cells treated with R9, but not in untreated cells. In addition, the formation of ceramide-enriched membrane platforms, was investigated (Fig 3). Using similar conditions as for ASMase detection, ceramide-enriched membrane platforms were detected in the HeLa cells treated with R9, but not in untreated cells. Effect of exogenous SMase activity on the threshold for peptide uptake via direct translocation's then investigated whether the concentration threshold for uptake via nucleation zones could be lowered by incubating HeLa cells with 5 μΜ R9, a concentration at which direct translocation is not observed, in the presence of a high concentration of bSMase. This experiment would provide information on whether the limiting factor for uptake via direct translocation is the peptide concentration or the induction of sphingomyelinase activity. As predicted, uptake of R9 through nucleation zones in the untreated cell population was virtually absent. In contrast, many cells with high fluorescence levels in the cytoplasm and nucleus were detected in the treated cell population (Fig. 4). Similar observations were made in mouse fibrosarcoma MC57 cells (data not shown). To assess the size limit for this sphingomyelinase-induced uptake of direct translocation, HeLa cells were incubated with R9 coupled to different cargos. In the presence of sphingomyelinase, we found an increased direct translocation of R9 conjugated to a short 7 amino acids apoptosis-enhancing smac-derived peptide derived from the proapoptotic smac protein though only at higher peptide concentrations as for R9. For R9 coupled to HPMA polymers no direct translocation was observed at concentrations up to 20 μΜ. Discussion

CPPs have a major potential for the delivery of membrane-impermeable molecules. To exploit this potential to the fullest, it is imperative that the import mechanisms and the associated effects on the cell are well understood. Despite various reports of direct translocation of arginine-rich CPPs and biophysical and molecular dynamics studies supporting this ability, the biology behind this mechanism, indicated by its sensitivity to various pharmacological interventions, has proven elusive. Here, we demonstrate that the previously poorly understood mechanism of direct translocation of cationic CPPs strongly depends on the induction of translocation of ASMase to the plasma membrane and ceramide formation.

We verified the involvement of ASMase activity in the rapid import mechanism a) by a pharmacological inhibition of ASMase using imipramine, b) by a rescue of the CPP uptake via exogenous addition of bSMase. The cellular events involved in the process, a translocation of ASMase to the outer leaflet of the plasma membrane and the formation of ceramide-enriched membrane platforms, were visualized by immunofluorescence. Moreover, we show that the threshold for direct translocation can be lowered by co-incubation of R9 with bSMase, which demonstrates that peptide concentration is not the limiting factor for uptake via this route, but it is in fact limiting for the induction of sphingomyelinase activity. To our knowledge, this is the first demonstration of a CPP-mediated induction of enzymatic activity that changes the composition in the plasma membrane in a way that stimulates CPP uptake. A schematic representation of the proposed import mechanism is presented in figure 5 A-C.

It still remains to be resolved, by which mechanism the cationic CPPs activate ASMase. It is known that, next to apoptosis-inducing growth factors, ASMase is activated by a variety of stressors However, the molecular mechanisms that link these stressors to activation have remained elusive. It is of note that reactive oxygen species have been implicated repeatedly in the activation of ASMase and that superoxide formation induced by octa-arginine was recently described in macrophages , although it is at present unclear whether these observations can be extended to other cell lines and CPPs.

The findings thus show that cationic CPPs do not function as inert delivery vectors, but have important biological effects instead. This evidently implies that their pharmacodynamic properties should be taken into account when designing CPP-based therapeutic strategies or when evaluating their outcomes. For instance, when multiple cationic CPP-drug conjugates were shown to induce ceramide, whereas the drugs did not do so by themselves , this activity was attributed to the mode of drug delivery rather than the vector itself. Our results imply that the bioactive properties of the cationic CPPs may have contributed to the enhanced cytotoxicity.

Since cationic CPPs can be expected to interact preferentially with cancer cell membranes due to their increased negative charge on the plasma membrane , it would be highly interesting to investigate whether cationic CPPs prefentially induce ceramide in tumor cells, which could lead to an enhancement of the specificity of tumor cell killing, when combined with chemotherapeutic drugs. Moreover, it is tempting to speculate on the possibility of a similar mechanism for cytotoxicity as observed for cationic antimicrobial peptide lacto ferritin B. This peptide, which kills tumor cells in a highly selective manner , accomplishes this through a ROS-dependent induction of apoptosis in many tumor cell lines, while leaving untransformed cells unaffected.

In conclusion, we show that the previously poorly understood direct cytoplasmic uptake of the cationic CPPs depends on a specifically induced enzymatic alteration of the composition of the plasma membrane, namely sphingomyelin cleavage to ceramide by ASMase. To our knowledge, no earlier studies have shown that delivery vectors can operate through actively changing the composition of the plasma membrane via an induced enzymatic activity. The finding that cationic CPPs by themselves induce ASMase is a further example of the notion that cationic CPPs do not act as true Trojan horses, i.e. purely pharmacokinetic modifiers, but have their own pharmacodynamic activity. Together with the induction of TNF receptor internalization , the activation of ASMase are two important factors that require scrutiny when designing applications.

Additional results depicted in figure 6

Cell cytotoxicity was measured with Resazurin Assay as described below. HeLa or Ovcar-3 cells (ATCC, Manassas, USA) were seeded at a density of 15xl0 ³ cells/well in a 96-well microtiter plate (Corning Incorporated, New York, USA) and incubated overnight in RPMI 1640 supplemented with 10 % (HeLa) or 20% (Ovcar-3) FCS. Ovcar-3 cells were incubated with varying concentrations of Taxol (Hospira Benelux BVBA, Brussel), whereas HeLa cells were incubated with varying concentrations of doxorubicin (Sigma- Aldrich), in the presence or absence of indicated concentrations of peptides. Resazurin is a non fluorescent dye which is chemically reduced by metabolically active live cells to highly fluorescent resorufm. Resazurin (Sigma- aldrich, Steinheim, Germany) was prepared fresh, 1 mg was dissolved in 1 mL

Phosphate Buffered Saline (lx) and diluted lOOOx in medium (RPMI 1640 with 10 % (HeLa) or 20% (Ovcar-3) FCS, 1 % pen/strep) before addition to the cells. The cells were incubated with the dye for 4 hours and fluorescence was measured in the Synergy 2 single channel microplate reader (BioTek Instruments, Inc., Winooski, USA).

We report in figure 6 that nona arginine, consisting either of L-amino acids (R9 or L- R9) or of D-amino acids (r9 or D-R9), sensitize cancer cells to the effects of chemotherapeutic agents when the drug and the peptides are added together as separate entities without any covalent coupling.

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