Patent Description:
A known side effect of many cytotoxic anticancer drugs is the development of Chemotherapy-induced peripheral neuropathy (CIPN). Clinical signs of CIPN include sensory loss, paresthesia, dysesthesia, numbness and tingling and often leads to neuropathic pain. Another known side effect is hair loss (alopecia).

Paclitaxel is a cytotoxic anticancer drug used in the treatment of solid organ tumours such as breast cancer and ovarian cancer. Paclitaxel is also used in adjuvant and neoadjuvant chemotherapy. While effective, paclitaxel provokes multiple side effects in patients. A common and potentially serious adverse reaction to treatment with paclitaxel is peripheral neuropathy, which occurs in up to <NUM>% of all patients. CIPN can be very serious, but even mild to moderate symptoms may persist for several years after treatment cessation, significantly impairing quality of life. With more than <NUM>% of breast cancer patients expected to be long-term survivors, this severe and long-lasting adverse reaction is particularly concerning and of public health interest. Further, treatment success with paclitaxel is correlated to dose and CIPN is the major reason for dose reduction, the development of CIPN may influence survival rates among cancer patients.

Hair loss (alopecia) is another known side effect of Paclitaxel. While less impactful on treatment success, hair loss presents a substantial psychological issue for cancer patients and survivors. Hair loss is also costly for the healthcare providers as wigs are needed for the patients.

Although described in higher detail for Paclitaxel above, other drugs or groups of drugs may also induce CIPN and/or hair loss (alopecia), such as other taxanes (e.g. docetaxel), vinca alkaloids (e.g. vincristine), epothilones (e.g. ixabepilone) and proteasome inhibitors (e.g. bortezomib).

<CIT> discloses the treatment of chemotherapy-induced peripheriral neuropathy (CIPN) derived from vincristine, etoposide and bortezumab by applying topical phenytoin. Thus, <CIT> relates to treatment of CIPN not prevention of side effects arising from chemotherapeutics, such as CIPN.

<CIT> reports on carbamazepine in combination with oxaliplatin for reducing CIPN derived from the anticancer agent. <CIT> is silent in respect of topically applying a P-gp inducer before administering a chemotherapeutic.

<CIT> relates to treatment of CIPN not prevention of side effects arising from chemotherapeutics, such as CIPN. <CIT> is silent in respect of topically applying P-gp inducer before administering a chemotherapeutic.

Thus, in order to increase quality of life and improve treatment outcomes among patients treated with certain cytotoxic anticancer drugs, methods for preventing or reducing side effects are needed.

Hence, improved methods for preventing and/or reducing side effects of chemotherapy (such as CIPN) would be advantageous, and in particular, a more efficient and/or reliable treatment regime for preventing CIPN or hair loss would be advantageous.

Embodiments not falling within the scope of the appended claims do not form part of the invention. Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

The present invention relates to the realization that dermal/topical application of a drug that induces P-gp can prevent side effects of chemotherapeutics which are transported out of cells by P-gp if applied before a chemotherapy treatment is initiated.

For example, the present invention describes that topical application (preferably to the hands and feet of a subject) of a drug that induces P-gp in the sensory neurons, can prevent CIPN if applied before a chemotherapy treatment is initiated.

Similar, the present invention discloses that topical application (preferably to the scalp, eyebrows, etc.) of a drug that induces P-gp can prevent alopecia, if applied before a chemotherapy treatment is initiated.

The application of the P-gp inducer prevents chemotherapy accumulation in the cells close to the skin surface (such as sensory neurons and hair follicles) by increasing the transport of chemotherapy out of the cells and therefore protects against local cell damage. This approach can be used for chemotherapeutics (e.g. vincristine, bortezomib and ixabepilone) that are transported out of cells by P-gp and which may cause e.g. peripheral neurotoxicity or alopecia.

Systemic induction of P-gp is undesirable, as this would lead to protection of tumours from chemotherapeutic drugs. However, local induction (e.g. by topical application) presents a novel mechanism to reduce accumulation of chemotherapy in e.g. sensory neurons and hair follicles that are damaged by chemotherapy.

Example <NUM> shows that the chemotherapeutic Paclitaxel affects neuronal morphology.

Example <NUM> shows that P-gp inhibition increases intracellular concentrations of paclitaxel (opposite effect of the present invention where an inducer is used).

Example <NUM> shows that P-gp inhibitors causes increased risk of peripheral neuropathy among patients treated with paclitaxel.

Example <NUM> shows that induction of P-gp and MRP1 with rifampicin protects cells from paclitaxel induced CIPN, thereby confirming the desired effect of the present invention.

Example <NUM> shows that inhibiting the efflux transporter MRP1 leads to substantially increased neurotoxicity of the chemotherapeutic vincristine (opposite effect of the present invention where an inducer is used).

Example <NUM> shows that induction of P-gp and MRP1 with rifampicin protects cells from vincristine induced CIPN, thereby confirming the desired effect of the present invention.

Example <NUM> shows that treatment with paclitaxel leads to increased expression of pain receptors and stress-factors in cells and that this response is alleviated in cells pretreated with rifampicin.

Example <NUM> shows that Rifampicin pretreatment leads to reduced intracellular accumulation of paclitaxel.

Example <NUM> shows that drugs that bind to and activate PXR (exemplified by carbamazepine and dexamethasone), increase expression of P-gp in sensory neurons derived from induced pluripotent stem cells.

Example <NUM> shows that rifampicin also increases expression of P-gp in other cells in the peripheral nervous system, namely Schwann cell precursors derived from induced pluripotent stem cells.

To summarize, the present invention relates to compositions comprising a P-gp inducer for topical use in preventing one or more side effects of chemotherapeutics, which chemotherapeutics are transported out of cells by P-gp.

Especially CIPN is a relevant side effect to treat/avoid, since CIPN may be a dose limiting side effect of chemotherapy. The invention also relates to kits and to cancer treatment of a subgroup of patients, which subgroup has been topically pretreated with a P-gp inducer,.

Thus, an object of the present invention relates to the provision of an improved treatment/prevention/lowering of CIPN.

Another object of the present invention relates to the provision of an improved treatment/prevention/lowering of Alopecia.

A further object, relates to an improved method for preventing one or more side effects of a chemotherapeutic and/or reducing the risk of getting one or more side effects of a chemotherapeutic and/or reducing the severity of one or more side effects of a chemotherapeutic and/or increasing the tolerability of a chemotherapeutic and/or removing dose limiting side effects of a chemotherapeutic in a subject.

In particular, it is an object of the present invention to provide a treatment/prevention regime that minimizes the risk of developing CIPN or lowers the side effects of CIPN.

An additional object relates to the provision of an improved treatment of cancer.

Another object is to provide a cancer treatment where the dose limit is higher compared to a cancer treatment without the improved treatment (addition of P-gp and/or MRP1 inducer). A dose limit is reached when a side effect of the chemotherapy is serious enough to prevent an increase in dose or level of the treatment.

Thus, one aspect of the invention relates to a composition comprising a P-gp inducer, for use in preventing one or more side effects of a chemotherapeutic and/or reducing the risk of getting one or more side effects of a chemotherapeutic and/or reducing the severity of one or more side effects of a chemotherapeutic and/or increasing the tolerability of a chemotherapeutic and/or removing dose limiting side effects of a chemotherapeutic in a subject,.

In a preferred embodiment, the side effect is CIPN.

Another aspect of the present invention relates to a kit of parts comprising.

Preferably the topical application is at least to the hands and feet in respect of CIPN and at least to the scalp when it comes to alopecia.

Still an aspect of the invention relates to a composition comprising a chemotherapeutic, which, in vivo, is transported out of cells by P-gp, for use in the treatment of cancer,.

The present invention will now be described in more detail in the following.

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:.

In the present context, "Chemotherapy-induced peripheral neuropathy" or "CIPN" refers to a progressive and enduring condition featuring pain, numbness, tingling and sensitivity to cold in the hands and feet (sometimes progressing to the arms and legs) that afflicts many patients undergoing chemotherapy. Chemotherapy-induced peripheral neuropathy (CIPN) is caused by many chemotherapeutic drugs, such as taxanes, vinca alkaloids, platins, bortezomib, ixabepilone and thalidomide.

The term "alopecia" refers to the partial or complete absence of hair from any area of the body where it normally grows. Chemotherapy-induced alopecia is most prominent on the scalp. Alopecia is a transient usually reversible side effect of many cancer chemotherapy treatments that can be psychologically devastating. The emotional trauma may be so severe as to lead to refusing or delaying treatment that might otherwise be beneficial. Recovery generally requires a period of several months to a year, amplifying the psychological impact of the disease and its treatment.

Several chemotherapeutics are known to induce chemotherapy-induced alopecia.

Paclitaxel is a cytotoxic anticancer drug used in the treatment of solid organ tumors such as breast cancer and ovarian cancer. Paclitaxel is also used in adjuvant and neoadjuvant chemotherapy. While effective, paclitaxel provokes multiple side effects in patients. A common and potentially serious adverse reaction to treatment with paclitaxel is peripheral neuropathy, which occurs in up to <NUM>% of all patients. Clinical signs of paclitaxel-induced peripheral neuropathy (PIPN) include sensory loss, paresthesia, dysesthesia, numbness and tingling and often leads to neuropathic pain. PIPN can be very serious, but even mild to moderate symptoms may persist for several years after treatment cessation, significantly impairing quality of life. With more than <NUM>% of breast cancer patients expected to be long-term survivors, this severe and long-lasting adverse reaction is particularly concerning and of public health interest. Further, treatment success with paclitaxel is correlated to dose and PIPN is the major reason for dose reduction, the development of PIPN may influence survival rates among cancer patients. Paclitaxel belongs to the class of chemotherapeutics named "taxanes" which are known to be transported out of cells by P-gp (<NPL>).

Bortezomib is used in the treatment of multiple myeloma (MM) and mantle cell lymphoma and is currently being tested in <NUM> clinical trials globally within indications such as MM, blood cancer and lymphomas.

Bortezomib belongs to the class of chemotherapeutics named "proteasome inhibitors", which are known to be transported out of cells by P-gp (<NPL>).

Vincristine is used in the treatment of many cancers, including leukemia, non-Hodgkin's lymphoma, thyroid cancer and brain tumors. Vincristine is widely used for treatment of childhood cancers and is currently being tested in <NUM> clinical trials globally within indications such as blood cancer, central nervous cancer (CNS) and various lymphomas. Vincristine belongs to the class of chemotherapeutics named "vinca alkaloids", which are known to be transported out of cells by P-gp (<NPL>).

Ixabepilone is a microtubule stabilizer, like paclitaxel, used in the treatment of aggressive metastatic or locally advanced breast cancer. Ixabepilone is a substrate for P-gp (<NPL>).

"P-glycoprotein" or "P-gp" or "Pgp" also known as multidrug resistance protein <NUM> (MDR1) or ATP-binding cassette sub-family B member <NUM> (ABCB1) or cluster of differentiation <NUM> (CD243) is an important protein of the cell membrane that pumps many foreign substances out of cells. It is an ATP-dependent efflux pump with broad substrate specificity. It exists in animals, fungi, and bacteria, and it likely evolved as a defence mechanism against harmful substances.

"Multidrug resistance-associated protein <NUM>" or "MRP1" is a protein that in humans is encoded by the ABCC1 gene. MRP1 is a member of the superfamily of ATP-binding cassette (ABC) transporters and is expressed in nearly all membranes in humans. ABC proteins transport various molecules across extra-and intra-cellular membranes. MRP1 is involved in multi-drug resistance.

In the present context, the term "P-glycoprotein (P-gp) inducer" refers to an agent that increases the expression of P-gp or increases the ATP-dependent efflux pump function of P-gp. Examples of P-gp inducers are rifampicin, carbamazepine and dexamethasone. P-gp expression is regulated by pregnane X receptor (PXR) and many inducers of P-gp are PXR agonists.

In the present context, the term "MRP1 inducer" refers to an agent that increases the expression of MRP1 or increases the transport function of MRP1. Examples of MRP1 inducers are rifampicin, carbamazepine and dexamethasone. MRP1 expression is regulated by pregnane X receptor (PXR) and many inducers of MRP1 are PXR agonists.

As outlined above, it has been found that topically applying a P-gp induce can help protect cells (locally) against damage from chemotherapeutics. Such protection is especially clinically relevant when it comes to CIPN, since it severely affects the health of the patient and may even be a dose limiting factor for the chemotherapeutic. However, local protection may also help in protection against less severe side effects such as hair loss and nail damage. Thus, an aspect of the invention relates to a composition comprising a P-gp inducer, for use in preventing one or more side effects of a chemotherapeutic and/or reducing the risk of getting one or more side effects of a chemotherapeutic and/or reducing the severity of one or more side effects of a chemotherapeutic and/or increasing the tolerability of a chemotherapeutic and/or removing dose limiting side effects of a chemotherapeutic in a subject,.

As outlined in the example section (see examples <NUM> and <NUM>), the addition of P-gp inducer prevents damage of the cells. This can e.g. be explored to protect e.g. against CIPN. Preferably, the inducer is applied topically to the skin, thus in an embodiment, said P-gp inducer is prepared for topical application to the subject. In the present context "topical administration" means application to body surfaces such as the skin, scalp and nail roots. In an embodiment, the composition is in the form of creams, foams, gels, lotions, and ointments. In another embodiment, the topical application is epicutaneous, meaning that it is applied directly to the skin.

CIPN is described in the example section, where the presence of P-gp in sensory neurons has been identified. It has been realized that this effect can be extrapolated to chemotherapy induced alopecia and nail root damage, which is often the consequence of chemotherapy.

In an embodiment, said P-gp inducer is for topical application to at least to the hands and/or feet and/or arms and/or legs, preferably at least to the hands and feet. CIPN is most often located to the hands and feet, but may also locate to arms and legs. Thus, these are the most relevant areas of the body to protect using topical application of the inducer. In the case of nail damage, the areas around the nails should be protected.

In an embodiment, the composition is for preventing CIPN and/or reducing the risk of developing CIPN and/or reducing the severity of CIPN in a subject.

The composition comprising the P-gp inducer may be further improved to reach the correct location under the skin. Thus, in an embodiment, the composition further comprising one or more enhancers that enhances that the P-gp inducer reaches the dermis of the cells.

In an embodiment, enhancers are selected from the group consisting of ethanol, acetone, glycols (e.g. polypropylene glycol), phosphatidylcholine and sodium lauryl sulphate. These enhancers may enhance that the P-gp reaches the dermis of the cells. Also, encapsulating the P-gp such as in liposomes, niosomes, emulsifiers, solid lipid nanoparticles or nanostructured lipid carriers may enhance reaching the dermis of the cells. Thus, in an embodiment, the P-gp inducer are encapsulated such as in liposomes, niosomes, emulsifiers, solid lipid nanoparticles and nanostructured lipid carriers.

In an embodiment, the composition comprises a pharmaceutically acceptable carrier, In one embodiment, the pharmaceutically acceptable carrier in the composition is a pharmaceutically acceptable carrier for topical use.

In one embodiment, the pharmaceutically acceptable carrier for topical use in the composition is a pharmaceutically acceptable carrier for topical use on the skin.

Thus, in an embodiment, the composition further comprises one or more enhancers that enhances that the P-gp inducer reaches the dermis of the cells.

In another embodiment, said P-gp inducer is prepared for topical application to the subject, at least to the scalp, eyebrows and/or eye lashes. Alopecia relates to hair loss, thus, for protection against hair loss regions with hair should be protected, such as hair on the head (such as to the scalp), the eye brows and eye lashes. Albeit protection against hair loss is at first glance of less clinical importance compared to CIPN, it is still damage to the cells and may be psychologically devastating to the patient. The emotional trauma may be so severe as to lead to refusing or delaying treatment that might otherwise be beneficial.

The inducers may work e.g. through inducing transporter activity or increasing expression. Thus, in an embodiment, the P-gp induces increased expression and/or increased activity of P-gp, preferably induces increased expression. P-gp and MRP1 are regulated by pregnane X receptor (PXR). Thus, agonists of PXR, such as rifampicin and carbamazepine will increase expression and thus activity of both efflux transporters.

The addition of inducers is only clinically relevant if the later used chemotherapeutics are transported out of the cells by P-gp.

Taxanes, vinca alkaloids, epothilones, anthracyclines, proteasome inhibitors and topoisomerase inhibitors are all chemotherapeutics, which are transported out of cells by P-gp.

Examples <NUM> and <NUM> show that the method of the invention works for a taxane (paclitaxel) and for a vinca alkaloid (vincristine).

In a further embodiment, the chemotherapeutic is selected from.

In yet an embodiment, the chemotherapeutic is selected from Docetaxel, paclitaxel, Vincristine, vinorelbine, Ixabepilone, Doxorubicin, daunorubicin, epirubicin, idarubicin, Etoposide, irinotecan, and topotecan; with the proviso that the composition is for preventing alopecia and/or reducing the risk of developing alopecia and/or reducing the severity of alopecia in a subject. These chemotherapeutics are all known to induce chemotherapy induced alopecia.

In yet a further embodiment, the chemotherapeutic is selected from Docetaxel, paclitaxel, Vincristine, Ixabepilone, and Bortezomib; with the proviso that the composition is for preventing CIPN and/or reducing the risk of developing CIPN and/or reducing the severity of CIPN in a subject. These chemotherapeutics are all known to induce CIPN.

In yet a further embodiment, the chemotherapeutic is selected from Paclitaxel, docetaxel and doxorubicin. These chemotherapeutics are all known to induce nail damage (nail root damage).

Different compounds are known to function as inducers of P-gp and/or MRP1.

Thus, in an embodiment, said P-gp inducer is selected from the group consisting of rifampicin, carbamazepine and dexamethasone. In e.g. examples <NUM> and <NUM> the P-gp /MRP1 inducer rifampicin has been tested for different types of chemotherapeutics.

P-gp inducers are carbamazepine, dexamethasone, doxorubicin, nefazodone, phenobarbital, phenytoin, prazosin, rifampicin, St. John's wort, tenofovir, tipranavir, trazodone, and vinblastine. This list of compounds includes common known pharmacological inducers of P-glycoprotein.

In yet another embodiment, said subject is scheduled for treatment with a chemotherapeutic selected from the group consisting of taxanes, vinca alkaloids, epothilones, antracyclines, proteasome inhibitors and topoisomerase inhibitors.

In a more specific embodiment relating to the composition for use,.

Example <NUM> shows the effect of rifampicin pretreatment in relation to paclitaxel treatment.

In another more specific embodiment relating to the composition for use,.

In yet another more specific embodiment relating to the composition for use,.

It is believed that there should be a certain period of time between (topically) applying the P-gp inducer and the chemotherapeutic. Thus, in embodiment, the P-gp inducer is prepared to be administered to the subject before administering the chemotherapeutic to the subject, such as prepared to be administered more than <NUM> day before administering the chemotherapeutic to the subject, such as more than <NUM> days, such as <NUM>-<NUM> days. The period may vary, thus (without being bound by theory) by having e.g. at least <NUM> day (<NUM> hours) between administration of the two compositions, there is time for the inducer to reach the cells, activate P-gp and be cleared from the body.

In an embodiment, the subject is a mammal, preferably a human, and more preferably a human suffering from cancer.

In an embodiment, the cancer is a solid organ tumor cancer such as breast cancer and ovarian cancer. Paclitaxel (a taxane) and epothilones is used for treatment of such cancers.

In another embodiment, the cancer is selected from the group consisting of blood cancer, multiple myeloma (MM), mantle cell lymphoma, and lymphomas. Bortezomib (a proteasome inhibitor) is used for treatment of such cancers.

In yet another embodiment, the cancer is selected from the group consisting of leukemia, non-Hodgkin's lymphoma, thyroid cancer, children's leukemia and brain tumors. Vincristine (a vinca alkaloid) is used for treatment of such cancers.

In an embodiment, the cancer is not skin cancer on the hands and/or feet. In another embodiment, the cancer is not Peripheral Nervous System (PNS) Cancer. It is to be understood that the cancer is not a cancer which primarily is located where the inducer is applied.

Since the present invention may involve both a P-gp inducer and a chemotherapeutic able to be transported by these efflux pumps, it could be relevant to have a kit comprising these components. Thus, an aspect of the invention relates to a kit of parts comprising.

The instructions may comprise instructions in relation to how and where to administer the compounds, concentrations and/or time between administering the components.

In an aspect, the invention also relates to the kit of parts according to the invention, for use as a medicament. As outlined above, the kit may be relevant in relation to side effects of chemotherapy such as CIPN, hair loss (Alopecia), and nail damage, such damage to the root of nails.

In another aspect, the kit of parts according to the invention is for use in the treatment or alleviation of a cancer in a subject. Albeit the invention relates to avoiding side effects of certain chemotherapies, the overall goal is treatment/alleviation of cancer. Thus, in an embodiment the invention relates to an improved treatment, namely fewer side effects. It could also allow the clinician to use higher concentrations of the chemotherapeutic thereby improving cancer treatment, while have the same amount of side effects as compared to treatment without the use of the inducer.

Thus, in an embodiment, the kit of parts is for use in preventing one or more side effects of chemotherapy and/or reducing the risk of getting one or more side effects of chemotherapy and/or reducing the severity of one or more side effects of chemotherapy, and/or increasing the tolerability of chemotherapy and/or removing dose limiting side effects of chemotherapy, in a subject,
wherein said one or more side effects is caused by a chemotherapeutic, which chemotherapeutic, in vivo, is transported out of cells by P-gp.

In yet an embodiment, said one or more side effects is selected from the group consisting of CIPN, hair loss (Alopecia), and nail damage, such as damage to the root of nails.

In yet another embodiment, said subject is considered at risk of developing CIPN, hair loss and/or nail damage.

As outlined above, the inducers are considered to be topically applied, preferably to the skin. Thus, in an embodiment, said P-gp inducer is prepared to be topically applied to the subject, preferably at least to the hands and/or feet and/or arms and/or legs, preferably at least to the hands and feet. These areas are particular relevant in relation to CIPN and nail damage.

In a related embodiment, said P-gp inducer inducer is prepared to be topically applied to the subject, preferably at least to the scalp, eyebrows and/or eye lashes. These areas are particular relevant in relation to hair loss.

The chemotherapeutic may be administered by any standard route for the specific drug. Thus, in an embodiment, the chemotherapeutic is prepared to be administered by a route selected from the group consisting of orally, intravenously, subcutaneous, intramuscular, intrathecal, and intraventricular.

The teaching of the present invention also allows for using known chemotherapeutics to treat a novel subgroup of patients. Thus, an aspect of the invention relates to composition comprising a chemotherapeutic, which, in vivo, is transported out of cells by P-gp, for use in the treatment of cancer,.

Topical treatment is described in further details for the other aspects of the invention. Thus, in an embodiment, the pretreatment is topical treatment at least to the hands and feet with the P-gp inducer and/or MRP1 inducer.

In another embodiment, the pretreatment is topical treatment at least to the scalp with the P-gp inducer and/or MRP1 inducer.

In a preferred embodiment, there is at least <NUM> hours between administering the P-gp inducers and/or MRP1 inducers and administering the chemotherapeutic.

In an embodiment, the chemotherapeutic is selected from the group consisting of taxanes, vinca alkaloids, epothilones, anthracyclines, proteasome inhibitors and topoisomerase inhibitors. These are chemotherapeutics, which, in vivo, are transported out of cells by P-gp and/or MRP1.

In yet an embodiment, the pretreatment with the P-gp inducer and/or MRP1 inducer is for preventing one or more side effects of the chemotherapeutic and/or reducing the risk of getting one or more side effects of the chemotherapeutic and/or reducing the severity of one or more side effects of the chemotherapeutic and/or increasing the tolerability of the chemotherapeutic and/or removing dose limiting side effects of the chemotherapeutic in the subject.

In a further embodiment, said one or more side effects is selected from the group consisting of CIPN, hair loss (Alopecia), and nail damage, such damage to the root of nails.

The composition may be administered to different location on the body,
depending on which side effect it to be avoided/minimized during the chemotherapy treatment. Thus, in an embodiment, the pretreatment with a P-gp inducer and/or MRP1 inducer is.

As outlined above, different P-gp inducers exist. Thus, in an embodiment, the P-gp inducer is selected from the group consisting of rifampicin, carbamazepine and dexamethasone.

It is considered important that a certain period of time is placed between the pretreatment the administration of the chemotherapeutic. The period may vary, thus (without being bound by theory) by having e.g. at least <NUM> day (<NUM> hours) between administration of the two compositions, there is time for the inducer to reach the cells, activate P-gp and be cleared from the body.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

The invention will now be described in further details in the following examples.

To evaluate how Paclitaxel affects neuronal morphology.

SH-SY5Y cells (<NUM>, Sigma-Aldrich) were kept in DMEM/F: <NUM> (<NUM>, ThermoFisher) with <NUM>% heat-inactivated fetal bovine serum (hiFBS, F9665 Sigma-Aldrich), <NUM>% penicillin/streptomycin (p/s) and <NUM> glutamine. Cells were split <NUM>:<NUM>-<NUM>:<NUM> using <NUM>% trypsin-EDTA (<NUM>, Fisher Scientific) at <NUM>-<NUM>% confluence. At high passage number, the epithelial cell type takes over the culture and causes drastically reduced efficiency of the differentiation. Thus, cells were only used below passage <NUM>.

Paclitaxel (T7402, Sigma-Aldrich) was diluted serially in DMSO (D2650, Sigma-Aldrich) and these stock solutions diluted <NUM>:<NUM> to ensure <NUM>% DMSO in all samples. These concentrations were selected based on the clinical pharmacokinetic profile of paclitaxel. Valspodar (SML0572, Sigma-Aldrich) were used at a final concentration of <NUM>. Antibody against TUBB3 (MA1-<NUM>, ThermoFisher Scientific) was used at <NUM>:<NUM>. Human dorsal root ganglion RNA for qPCR analysis was purchased from Clontech Laboratories (<NUM>, Clontech laboratories, Inc, Mountain View, California, USA). Human dorsal root ganglion for protein quantification of ABCB1 was obtained from National Disease Research Interchange (Philadelphia, PA, USA).

SH-SY5Y cells were differentiated as previously described in detail by <NPL>). Briefly, SH-SY5Y cells below passage <NUM> were seeded at <NUM>-<NUM> x10<NUM> cells/cm<NUM> in uncoated <NUM>-well plates (day <NUM> (D0)) and left overnight in growth media. The next day (D1) differentiation was initiated using DMEM/F:<NUM> media with <NUM>% hiFBS, <NUM>% p/s, <NUM> glutamine and <NUM> retinoic acid (RA, R2625 Sigma-Aldrich) and media was replaced at D3 and D5. On D7 cells were split <NUM>:<NUM> using <NUM>% trypsin-EDTA to uncoated dishes and on D8 hiFBS content was reduced to <NUM>%. On D10 the cells were split <NUM>:<NUM> to extracellular matrix-coated dishes (E0282, Sigma-Aldrich). On D11 media was replaced with the final differentiation media containing neurobasal media (<NUM>, ThermoFisher) with 1X B27 supplement (<NUM>, ThermoFisher), <NUM> KCI (<NUM>, Fisher Scientific), <NUM>% p/s, 1X Glutamax, <NUM> ng/ml brain-derived neurotrophic factor (BDNF, CST-<NUM>, Peprotech), <NUM> dibutyryl cyclic AMP (db-cAMP, D0627 Sigma-Aldrich) and <NUM> RA. Media was replaced on D14 and D17 and cells were tested and confirmed as fully differentiated on D18 and used for downstream applications as outlined below.

Total RNA from SH-SY5Y cell lines was isolated using an RNeasy Mini kit (Qiagen, Valencia, CA). Total RNA (<NUM>µg) was reverse transcribed into cDNA using a SuperScript VILO cDNA Synthesis kit (Life Technologies, CA). Quantitative real-time PCR was carried out in <NUM>-well reaction plates using 2X Taqman Fast Universal Master Mix (Applied Biosystems, Foster City, CA), 20X Taqman specific gene expression probes for each transporter, and <NUM> ng of the cDNA template. The reactions were carried out on an Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The relative expression level of each mRNA transcript was calculated by the comparative method (ΔCt method), normalized to the housekeeping gene, hypoxanthine phosphoribosyl transferase (HPRT).

Both differentiated and undifferentiated SH-SY5Y cells were fixed using <NUM>% paraformaldehyde. Cells were permeabilized using <NUM>% Triton X-<NUM> and unspecific binding was blocked using bovine serum albumin (BSA). Primary antibodies were incubated overnight at <NUM>OC and secondary antibodies were incubated for one hour at room temperature. Nuclei were stained using <NUM> DAPI for <NUM> minutes. Imaging was performed using a Leica DMI4000B microscope. Neurite morphology was assessed using the ImageJ modules CellCounter and Simple Neurite Tracer. To determine number of neurites per cell, CellCounter was used to count the number of cells with <NUM>, <NUM>, <NUM>. n neurites. Neurite length was determined using Simple Neurite Tracer. For both endpoints, at least five images of each condition from three separate differentiations were used. ImageJ analysis was blinded to the person assessing length and number of neurites to ensure no bias.

Fully differentiated SH-SY5Y cells were exposed to <NUM>-<NUM> paclitaxel for <NUM> hour with and without <NUM> hour pre-incubation with efflux transporter inhibitors. After this, media was aspirated and cells were lysed using radioimmunoprecipitation assay (RIPA) buffer with <NUM>% protease inhibitor for <NUM> minutes on ice. Cells were collected using a cell scraper, vortexed and sonicated (<NUM> sec X2), centrifuged and the supernatant was stored at -<NUM>OC until LC-MS/MS analysis. The concentration of paclitaxel in the cell lysate was determined at the Department of Clinical Pharmacology and Pharmacy, Institute of Public Health, University of Southern Denmark by use of liquid chromatography and tandem mass spectrometry (LC-MS/MS). The LC-MS/MS system consisted of an Ultimate <NUM> UHPLC system connected to a TSQ Quantiva Triple Quadropole Mass Spectrometer with heated electrospray ionization (H-ESI) (Thermo Scientific, San José, CA). The ionization was performed in positive mode with a spray voltage of <NUM> V, sheath gas <NUM> (AU), aux gas <NUM> (AU), sweep gas <NUM> (AU) and a vaporizer temperature of <NUM>. The ion transfer tube temperature was <NUM>. Data acquisition was performed in single reaction monitoring (SRM) mode. Paclitaxel was quantified at the transition from (m/z) <NUM> - <NUM>, and with (m/z) <NUM> - <NUM> and (m/z) <NUM> - <NUM> as qualifier traces. The analytical separation was performed by use of a Hypersil GOLD (C18) <NUM> x <NUM> (<NUM>) column (Thermo Scientific, San José, CA) with a mobile phase of acetonitrile: <NUM> formic acid (<NUM>:<NUM> v/v) at a flow rate of <NUM>/min. The sample preparation of the cell lysate consisted of a single dilution step. A volume of <NUM>µL cell lysate and <NUM>µL acetonitrile was pipetted into a <NUM> polypropylene micro tube (Sarsted, Nürnbrecht, Germany). The sample was vortex mixed for <NUM>, and thereafter centrifuged at <NUM>,<NUM> g for <NUM>. A volume of <NUM>µL of the supernatant was injected onto the LC-MS/MS system. Calibration curves ranging from <NUM> to <NUM> as well as quality control samples were prepared and included in each batch of analysis. The intra- and interday variability was < <NUM>%. The limit of detection (LOD) for the method was <NUM> ng/mL and limit of quantification (LOQ) was <NUM> ng/mL. Finally, paclitaxel concentrations were adjusted for protein concentration in each well as assessed using a Pierce BCA protein assay kit.

Transporter protein expression levels were analyzed by TXP targeted proteomic analysis, which has been described before. In short, cell pellets were incubated for one hour with lysis buffer containing <NUM>% NP-<NUM> (Thermo Fisher), <NUM>% SDS (ThermoFisher), <NUM> NaCl (Merck), <NUM> di-Sodium hydrogen phosphate dihydrate (Merck), <NUM> EDTA, and <NUM> units/mL Benzonase (Novagen). The protein concentration in the lysate was determined by BCA assay (Thermo Fisher Scientific) according to the manufacturer's manual. Subsequently, <NUM>µg protein was proteolyzed with trypsin overnight. Stable isotope labelled peptides and TXP-antibodies (customized produced by Pineda) were added to <NUM> or <NUM>µg of the digest and incubated for one hour. Peptide -antibody complexes were precipitated, washed and denatured by using protein G-coated magnetic beads (ThermoFisher) and a magnetic particle processor (KingFisher <NUM>, ThermoFisher). The precipitated peptides were subsequently quantified using the previously described <NUM> LC-MS method (UltiMate <NUM> RSLCnano and tSIM - QExactive Plus™ Thermo Scientific, Waltham, USA) (<NPL>). LC gradients were optimized for each multiplex. Raw data were processed with Skyline v4. Peak areas of isotopically-labeled peptides representing known peptide amounts and endogenous signals were set in relation to one another on parent ion level.

Analysis of variance (ANOVA) was used to assess dose-dependent effects of paclitaxel on number and length of neurites and intracellular accumulation of paclitaxel. Data were log-transformed to ensure normality of data. STATA software version <NUM> was used to perform the statistical analysis. The logistic regression to assess risk of dose modification of paclitaxel due to P-gp inhibitors was adjusted for age, body surface, tumor type, cancer stage, treatment schedule and previous chemotherapy. SPSS software version <NUM> was used to perform the logistic regression analysis.

SH-SY5Y cells were effectively differentiated to neurons using the protocol of <NPL>). After <NUM> days of differentiation the cells form a complex neuronal network with high TUBB3 expression and the majority of cells (><NUM>%) were negative for the proliferation marker Ki67, indicating that the cells are post-mitotic (data not shown).

Neuronal morphology was assessed by measuring neurite length and the number of neurites/cell in fully differentiated SH-SY5Y cells with <NUM> hours paclitaxel treatment. Paclitaxel induced a dose-dependent neurotoxicity to differentiated SH-SY5Y cells and significantly reduced both the number of neurites (p<<NUM>) and their length (p<<NUM>) (<FIG>).

mRNA and protein expression of relevant drug transporters were assessed by qPCR and LC-MS/MS and indicated expression of the efflux transporters P-gp and MRP1 in both SH-SY5Y cells and human dorsal root ganglion (data now shown). To assess the role of P-gp on the neuronal accumulation and neurotoxicity of paclitaxel, P-gp was inhibited with <NUM> valspodar. Inhibiting P-gp with valspodar substantially exacerbated the neuronal toxicity of paclitaxel as indicated by a decrease in the number and length of neurites (ANOVA p<<NUM>, <FIG> and <FIG>). Valspodar did not affect the neuronal morphology without paclitaxel present (<FIG>).

Inhibiting P-gp increased the neuronal toxicity of paclitaxel in SH-SY5Y neuron cells. These data also suggests that stimulating P-gp would have the opposite effect and thus also that CIPN could be prevented by applying such P-gp inducers to the skin.

To confirm that the increased harmful effect of paclitaxel on neuronal morphology under conditions of P-gp inhibition is related to increased exposure, the intracellular concentrations of paclitaxel were quantified.

Cells were treated with paclitaxel with and without concomitant P-gp inhibition by valspodar and intracellular levels were measured <NUM> hour after treatment.

For further details, see also materials and methods section in example <NUM>.

Inhibition of P-gp caused a ><NUM>-fold increase in intracellular accumulation of paclitaxel at <NUM> (p<<NUM>, <FIG>).

P-gp inhibition causes increased accumulation of paclitaxel in SH-SY5Y-derived neurons. Again, these data suggest that stimulating P-gp would have the opposite effect and thus also that CIPN could be prevented by applying such P-gp inducers to the skin.

To assess if concomitant ingestion of P-gp inhibitors causes increased risk of peripheral neuropathy among patients treated with paclitaxel.

To assess if P-gp inhibition leads to increased risk of peripheral neuropathy we used a previously described database of <NUM> paclitaxel treated patients with breast or ovarian cancer (<NPL>). Briefly, dose-modifications due to peripheral neuropathy were collected from medical records and multivariate logistic regression was performed to assess if users of P-gp inhibitors had a higher risk of dose modifications due to neuropathy. P-gp inhibitors were selected based on two literature reviews (see comments under table <NUM>). From these reviews, we identified drugs listed as P-gp inhibitors (drugs classified as inhibitor in either review), and, within these, strong P-gp inhibitors (drugs classified as inhibitor in both reviews).

The association from univariate and multivariate analysis between paclitaxel dose modification due to peripheral neuropathy and concomitant P-gp inhibitor use is shown in Table <NUM>.

Patients treated with any P-gp inhibitor had a <NUM>-fold (<NUM>% confidence interval (CI): <NUM>-<NUM>) increased risk of dose modification due to peripheral neuropathy, patients treated with strong inhibitors led to <NUM>-fold (<NUM>% CI: <NUM>-<NUM>) higher risk and patients treated with atorvastatin had <NUM>-fold (<NUM>% CI: <NUM>-<NUM>) increased risk, while users of simvastatin was not at increased risk.

These data clearly indicates that systemically administered P-gp inhibitors increase the risk of CIPN in patients treated with paclitaxel. Again, these data suggest that stimulating P-gp would have the opposite effect and that CIPN could be prevented by applying such P-gp inducers to the skin.

To verify that induction of P-gp with rifampicin protects neurons from paclitaxel induced peripheral neuropathy.

Cells were pretreated for <NUM> hours with <NUM> rifampicin prior to addition of paclitaxel treatment.

Low concentrations of rifampicin significantly increases the expression of the P-gp drug transporter (Figure 4A) and reduces accumulation of paclitaxel by <NUM>-fold (Figure 4B). Higher expression of P-gp protects neuron from paclitaxel damage (<FIG>).

These data clearly show that by adding a drug that increases the expression of drug transporters, the intracellular concentration of the chemotherapeutical paclitaxel is lowered.

These results directly support that by topically applying a P-gp inducer (and MRP1 inducer) to the skin, the amount of intracellular chemotherapeutic in the skin neurons can be lowered, and thereby also lowering the risk that the patient develops side effects of the chemotherapy, such as CIPN, while still maintaining the full effect of the chemotherapeutical at relevant location in the body (the cancer cells).

Further, since P-gp and MRP1 are known to be expressed in hair follicles (<NPL>), the same effect can be obtained for these cell types, thereby protecting the patient from hair loss.

To determine vincristine toxicity in sensory neurons and determine the impact of inhibiting the efflux transporter multidrug resistance-associated protein <NUM> (MRP1).

Sensory neurons were generated from human induced pluripotent stem cells (iPSCs) by implementing the well-established protocol by Chambers et. (Nat Biotech <NUM>). The neuronal differentiation was initiated when iPSCs achieved <NUM>-<NUM>% confluency using KSR medium, which contained <NUM>% KnockOut DMEM (Gibco, <NUM>-<NUM>) and <NUM>% KnockOut Serum Replacement (KSR, ThermoFisher, <NUM>-<NUM>), <NUM>% GlutaMAX-I (Gibco, <NUM>-<NUM>), <NUM>% mini- mum essential medium non-essential amino acids (MEM-NEAA, Gibco, <NUM>-<NUM>), <NUM>% P/S and <NUM> β-mercaptoethanol (Gibco, <NUM>).

On days <NUM>-<NUM>, SMAD signaling was inhibited by adding <NUM> LDN-<NUM> (Selleck, S7507) and <NUM> SB-<NUM> (Selleck, S1067) to the medium. Medium was changed daily, and N2 medium was added with <NUM>% increments every other day starting on day <NUM> (<NUM>% N2 on day <NUM>). N2 medi- um consists of <NUM>% Neurobasal medium (Gibco, <NUM>-<NUM>) with <NUM>% N2 Supplement (Gibco, <NUM>-<NUM>), <NUM>% B27 Supplement (Gibco, <NUM>-<NUM>) and <NUM>% P/S. On days <NUM>-<NUM>, nociceptor induc- tion was initiated with the addition of the three inhibitors, <NUM> CHIR99021 (Selleck, S1263), <NUM> SU5402 (Selleck, S7667) and <NUM> DAPT (Selleck, S2215).

For long-term cultivation starting on day <NUM>, cells were washed with PBS÷ and incubated with Accutase for <NUM> to obtain a single-cell suspension. After incubation, the culture plates were gently rocked and cellular clumps were mechanically dissociated by pipetting up and down. The cells were filtered through a <NUM> nylon cell strainer (Corning, <NUM>) and centrifuged (<NUM> rpm, <NUM>, RT). Following resuspension of the pellet, iPSC-SNs were seeded as single cells on PLO/LAM/FN- coated culture plates at a density of <NUM>,<NUM> viable cells/cm<NUM>. The long-term cultivation medium contained <NUM>% N2 medium supplemented with <NUM> ng/mL nerve growth factor β (NGF-β, PeproTech, <NUM>-<NUM>), brain-derived neurotrophic factor (BDNF, PeproTech, AF450-<NUM>), glial cell line- derived neurotrophic factor (GDNF, PeproTech, AF450-<NUM>), neurotrophin <NUM> (NT-<NUM>, PeproTech, <NUM>- <NUM>) and <NUM> ascorbic acid (AA, Sigma, A4403).

The medium was changed every fifth day by gently removing half of the medium and replacing it with fresh long-term cultivation medium. To reduce the non-neuronal population, cells were treated with <NUM>µg/mL Mitomycin C (Sigma, M4287) for <NUM> hours on day <NUM>. Once a week, laminin was added to the medium to maintain cell attachment.

Cells were treated with vincristine from <NUM>-<NUM> for <NUM> hours and immunolabeled with peripherin antibody. Quantification of network was done using ImageJ and MIPAR. In inhibition experiments, cells are pretreated with <NUM> MK-<NUM> for one hour ahead of chemotherapy treatment.

Vincristine (VCR) leads to substantial neurotoxicity in iPSC-derived sensory neurons. Thus, vincristine causes a dose-dependent decrease in number of neurites extending from a cluster of sensory neurons (<FIG>). Additionally, neurite length is shortened in a dose-dependent manner (<FIG>).

Inhibiting the efflux transporter MRP1 with MK-<NUM> (MK) leads to substantially increased neurotoxicity even at low vincristine concentrations that are otherwise not toxic (<FIG>).

These data indicate that vincristine causes substantial neurotoxicity in iPSC-derived sensory neurons. Additionally, inhibition of the efflux transporter MRP1 was shown to exacerbate vincristine-induced neurotoxicity.

These data also suggest that stimulating MRP1 would have the opposite effect and thus also that e.g. CIPN could be prevented by applying such MRP1 inducers to the skin.

To determine if rifampicin protects against vincristine-induced peripheral neuropathy.

Induced pluripotent stem cells were differentiated to sensory neurons for <NUM>-<NUM> days as described above. Rifampicin was added <NUM> hours ahead of vincristine to increase expression of efflux transporters.

Vincristine-induced neurotoxicity was substantially alleviated with pretreatment with <NUM> rifampicin (<FIG>).

Rifampicin (RIF) protects against vincristine (VCR)-induced neurotoxicity likely through upregulation of the efflux transporter MRP1.

The aim of this study was to assess the transcriptional impact of chemotherapy treatment on the stress-related protein ATF-<NUM> and the pain-sensing receptor TRPV1.

Induced pluripotent stem cells were differentiated to sensory neurons for <NUM>-<NUM> days as described above. Rifampicin was added <NUM> hours ahead of paclitaxel to increase expression of efflux transporters. Expression levels was determined using real-time qualitative polymerase chain reaction (RT-qPCR) for TRPV1 and ATF-<NUM>.

Paclitaxel (PTX) causes substantially increased expression of both TRPV1 (<FIG>) and ATF-<NUM> (<FIG>) in a dose-dependent manner. Pretreatment with rifampicin (RIF) protects against these transcriptional changes.

Rifampicin-induced upregulation of P-gp leads to lower intracellular concentrations of paclitaxel leading to protection against transcriptional changes caused by paclitaxel. This supports the claim that P-gp induction should protect against CIPN through limiting the intracellular concentrations of paclitaxel. Similar mechanisms are expected to limit chemotherapy disposition to hair follicles and thus protecting against alopecia.

To determine intracellular concentrations of paclitaxel in iPSC-derived sensory neurons and to assess if rifampicin pretreatment leads to lower accumulation.

Induced pluripotent stem cells were differentiated to sensory neurons for <NUM>-<NUM> days as described above. Rifampicin was added <NUM> hours ahead of paclitaxel to allow sufficient time to increase expression of efflux transporters. One and <NUM> paclitaxel was added to the cells for <NUM> hour and the cells were lysed using RIPA buffer. Paclitaxel concentrations was determined using an already established LC-MS/MS method.

Intracellular concentrations of paclitaxel were approximately <NUM>-fold lower after pretreatment with rifampicin (<FIG>).

Rifampicin pretreatment leads to reduced accumulation of the chemotherapeutic paclitaxel causing reduced neuronal toxicity as described also above.

To confirm that additional PXR agonists, besides rifampicin, increase expression of P-gp in sensory neurons derived from induced pluripotent stem cells.

Induced pluripotent stem cells were differentiated to sensory neurons for <NUM>-<NUM> days as described above. Cells were treated with carbamazepine and dexamethasone for <NUM> hours. Expression level of P-gp (ABCB1) was determined using real-time qualitative polymerase chain reaction (RT-qPCR) and adjusted for a housekeeping gene (GAPDH). Data are shown as relative expression to control (<NUM>% DMSO).

Both carbamazepine and dexamethasone increase expression of P-gp by up to <NUM>% (<FIG>).

This example shows that drugs that activate PXR (exemplified by carbamazepine and dexamethasone), increase expression of P-gp in sensory neurons derived from induced pluripotent stem cells. This supports the claim that PXR agonists will be able to protect against chemotherapeutics that are substrates of P-gp such as paclitaxel, vincristine, bortezomib and ixabepilone by increasing expression of P-gp and reducing accumulation of these drugs in sensory neurons.

The aim of this study was to assess if rifampicin increases expression of P-gp in Schwann cells. Schwann cells are the principal glial cell in the peripheral nervous system. Schwann cells exist as both myelinating and non-myelinating and myelinating Schwann cells ensheath large-diameter neurons in layers of myelin, thus increasing conductance. Non-myelinating Schwann cells support neurons by secreting neuronal growth factors that play a crucial role in maintaining and repairing neurons following damage and may transmit pain signalling.

Induced pluripotent stem cells (iPSC) were differentiated into Schwann cell precursors using a previously published protocol. iPSC were plated onto growth factor-reduced Matrigel-coated culture dishes. The next day, the culture medium was switched from iPSC culture medium to neural differentiation medium (NDM) containing 1x N2, 1x B27, <NUM>% BSA, <NUM> GlutaMAX, <NUM> β-mercaptoethanol, <NUM> CT <NUM>, and <NUM> SB431542 in advanced DMEM/F12 and Neurobasal medium (<NUM>:<NUM> mix). After <NUM> days of differentiation, the medium was replaced with neural induction medium containing <NUM> ng/mL NRG1 (this is designated Schwann cell precursor differentiation medium [SCPDM]) and replaced with fresh medium daily. The cells were routinely dissociated with Accutase treatment upon reaching <NUM>% confluence and were expanded by additional cultivation in SCPDM. The hPSC-derived SCPs were generated after approximately <NUM> days of differentiation. SCPDM was used for the induction and maintenance of iPSC-derived Schwann cell precursors.

Schwann cell precursors were treated with rifampicine for <NUM> hours. Expression level of P-gp (ABCB1) was determined using real-time qualitative polymerase chain reaction (RT-qPCR) and adjusted for a housekeeping gene (GAPDH). Data are shown as relative expression to control (<NUM>% DMSO).

Expression of P-gp was increased in a dose-dependent manner in Schwann cell precursors derived from iPSC (<FIG>).

Claim 1:
A composition comprising a P-gp inducer, for use in preventing one or more side effects of a chemotherapeutic and/or reducing the risk of getting one or more side effects of a chemotherapeutic and/or reducing the severity of one or more side effects of a chemotherapeutic and/or increasing the tolerability of a chemotherapeutic and/or removing dose limiting side effects of a chemotherapeutic in a subject,
wherein, said chemotherapeutic, in vivo, is transported out of cells by P-gp;
wherein the P-gp inducer is selected from the group consisting of rifampicin, carbamazepine and dexamethasone,
wherein the chemotherapeutic is selected from the group consisting of taxanes, vinca alkaloids, epothilones, anthracyclines, proteasome inhibitors and topoisomerase inhibitors;
wherein the P-gp inducer is prepared to be topically administered to the subject before administering the chemotherapeutic to the subject; and
wherein said one or more side effects is selected from the group consisting of CIPN (i.e. chemotherapy-induced peripheral neuropathy), hair loss (alopecia), and nail damage, such as damage to the root of nails.