NOVEL DRUG PRODUCTS OF PICROPODOPHYLLIN

FIELD OF THE INVENTION

The present invention is directed to solid, stabilized picropodophyllin which is substantially XRPD amorphous, as well as to a method of preparing the same. The invention is furthermore directed to a pharmaceutical formulation comprising said solid, stabilized amorphous picropodophyllin, and its use in therapy such as in cancer therapy.

BACKGROUND OF THE INVENTION

One of the largest challenges in pharmaceutical drug development is that drug compounds often are poorly soluble, or even insoluble, in aqeous media. Insufficient drug solubility means insufficient bioavailability, as well as poor plasma exposure of the drug when administered to humans and animals. Variability of plasma exposure in humans is yet a problem when developing drugs which are poorly soluble, or even insoluble, in aqeous media.

It is estimated that between 40% and 70 % of all new chemical entities identified in drug discovery programs, are insufficiently soluble in aqeous media (M. Lindenberg, S et al: European Journal of Pharmaceutics and Biopharmaceuticals, vol. 58, no.2, pp. 265-278, 2004). Scientists have investigated various ways of solving the problem with poor drug solubility in order to enhance bioavailability of poorly absorbed drugs, aiming at increasing their clinical efficacy when administered orally.

Technologies such as increase of the surface area and hence dissolution may sometimes solve solubility problems. Other techniques that may also solve bioavailability problems are addition of surfactants and polymers. However, each chemical compound has its own unique chemical and physical properties, and hence has its own unique challenges when being formulated into a pharmaceutical product that can exert its clinical efficacy.

Picropodophyllin is an insulin-like growth factor-1 receptor inhibitor fiGF-lR inhibitor) small- molecule compound belonging to the class of compounds denominated cyclolignans, having the chemical structure:

The patent applicant is presently entering clinical phase II development with its development compound picropodophyllin (AXL1717). However, picropodophyllin is poorly soluble in aqueous media. In a phase I clinical study performed by the applicant in 2012 (Ekman S et al; Acta Oncologica, 2016; 55: pp. 140-148), it was discovered that picropodophyllin, when administered as an oral suspension to lung cancer patients, resulted in unacceptable variability in drug exposure. A large variability in plasma exposure of the active drug picropodophyllin occurred not only within certain patients, but also between several patients.

Yet a problem with administering picropodophyllin as an aqeous solution, is that due to the poor solubility in aqueous media, it is difficult or even impossible to reach the required therapeutic doses.

The compound picropodophyllin is furthermore physically unstable, and transforms from amorphous picropodophyllin into crystalline picropodophyllin. Yet a stability problem with picropodophyllin is that it is chemically unstable in solution.

DESCRIPTION OF THE INVENTION

An aspect of the present invention is to provide solid, stabilized picropodophyllin which is substantially XRPD amorphous, comprising:

(i) picropodophyllin; and

(ii) at least one water-soluble polymer selected from FDA approved GRAS polymers; wherein

(a) the amount of picropodophyllin is 20 %-80 % by wt; and

(b) the total amount of water-soluble polymer is 20 %-80 % by wt. Picropodophyilin is an insulin-like growth factor-1 receptor inhibitor (!GF-IR inhibitor) small- molecule compound belonging to the class of compounds denominated cyclolignans, having the chemical structure:

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the amount of picropodophyilin is 30% - 60% by wt.

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the amount of picropodophyilin is 40% - 50% by wt.

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the amount of picropodophyilin is 20 %, or 30 %, or 40 %, or 50 %, or 60 %, or 70 %, or 80%, by weight.

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the total amount of water-soluble polymer is 40%-70% by wt.

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the total amount of water-soluble polymer is 50%-60% by wt.

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the total amount of water-soluble polymer is 20 %, or 30 %, or 40 %, or 50 %, or 60 %, or 70 %, or 80%, by weight.

An aspect of the present invention is to provide solid, stabilized picropodophyilin as herein described and claimed, wherein the water-soluble polymer is selected from hydroxypropylmethyl cellulose (HPMC); hydroxypropyl cellulose (HPC); polyvinylpyrrolidone (PVP); methyl cellulose (MC);

polyethylene glycol (PEG); polyvinyl alcohol (PVA); polyacrylic acid (PAA);

N-(2-hydroxypropyl)methacrylamide (HPMA); hydroxyethyl cellulose (HEC);

hydroxyethylmethyl cellulose(HEMC); carboxymethylethyl cellulose; sodium carboxymethyl cellulose; potassium carboxymethyl cellulose; copolymers of vinylpyrrolidone; vinylpyrrolidone-vinylacetate copolymer (copovidone); copolymers of vinyl acetate; copolymers of vinyl propionate; copolymers of vinyl acetate and crotonic acid; partially hydrolyzed polyvinyl acetate; gelatin; sodium alginate;

soluble starch; gum acacia; dextrin; hyaluronic acid; sodium chondroitin sulfate; propyleneglycol alginate; agar; tragacanth; xanthan gum; polyvinyl-acetal-diethylaminoacetate; macrogol;

polyethylene oxide; polypropylene oxide; copolymers of ethylene oxide (EO) and

propylene oxide (PO); carrageenans; galactomannans; or a mixture of at least two or more of said water-soluble polymers.

In one aspect of the invention, the water-soluble polymer is hydroxypropyl methyl cellulose (HPMC) or polyvinyl pyrrolidone (PVP).

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, comprising a mixture of two or more water-soluble polymers.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 1:1.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 1:2.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 1:3.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 1:4. An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 1:5.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 2:3. An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 2.5:1.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 3:2.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 4:1.

An aspect of the present invention is to provide solid, stabilized picropodophyllin as herein described and claimed, wherein the ratio of picropodophyllin and the at least one water-soluble polymer is 7:3.

An aspect of the present invention is a pharmaceutical formulation comprising solid, stabilized picropodophyllin as herein described and claimed, optionally in admixture with one or more pharmaceutically and pharmacologically acceptable excipients, carriers and/or fillers.

FIGURES

Figure 1 shows an XRPD pattern of picropodophyllin monohydrate (crystalline material).

Figure 2 shows an XRPD pattern of amorphous picropodophyllin prepared according to Example 1. Figure 3 shows an XRPD pattern of amorphous picropodophyllin prepared according to Example 2. Figure 4 shows an XRPD pattern of amorphous picropodophyllin prepared according to Example 3. Figure 5 shows an XRPD pattern of amorphous picropodophyllin prepared according to Example 4. Figure 6 shows an XRPD pattern of amorphous picropodophyllin prepared according to Example 5. Figure 7 shows an XRPD pattern of amorphous picropodophyllin prepared according to Example 6. Figure 8 shows an XRPD pattern of picropodophyllin prepared according to Example 7.

Figure 9 shows an XRPD pattern of picropodophyllin (crystalline material, indicated with the number 3 in this figure) superimposed on an XRPD pattern for amorphous picropodophyllin prepared according to Example 4 (indicated with the number 1 in this figure) and Example 6 (indicated with the number 2 in this figure) respectively.

Figure 10A, B and C shows an XRPD pattern of amorphous picropodophyllin prepared according to Examples 11A, 11B and 11C.

Figure 11A and B shows an XRPD pattern of amorphous picropodophyllin prepared according to Examples 12A and 12B. Figure 12A and B shows an XRPD pattern of amorphous picropodophyllin prepared according to Examples 13A and 13B.

Figure 13 A, B and C shows an XRPD pattern of picropodophyllin drug product prepared according to Examples 11A, 11B and 11C. The XRPD pattern at start (0 months) is shown on top, and the XRPD pattern at 3 months is shown at the bottom.

Figure 14A and B shows an XRPD pattern of picropodophyllin drug product prepared according to Examples 12A and 12B. In Figure 14A, the XRPD pattern at start (0 months) is shown on top, and the XRPD pattern at 3 months is shown at the bottom.

Figure 15A and B shows an XRPD pattern of picropodophyllin drug product prepared according to Examples 13A and 13B. The XRPD pattern at start (0 months) is shown on top, and the XRPD pattern at 3 months is shown at the bottom.

DEFINITIONS

The wording "picropodophyllin" as used herein, is defined as a compound of formula I:

The wording "poorly soluble in aqueous media" is herein defined as picropodophyllin having a solubility in water of less than or equal to 30 pg/ml, such as 10-30 pg/ml.

In one aspect of the present invention, picropodophyllin as herein described and claimed, has a solubility in aqeous media which is in the mg/ml magnitude.

The wording "amorphous" as used herein, refers to a solid material which does not show the characteristics of a crystalline material, and which instead of sharp peaks shows one or more broad peak(s) in an XRPD diffractogram. The wording "crystalline" means that the structural units of an active pharmaceutical agent (API) are arranged in fixed geometric pattern or lattices, so that crystalline solids have rigid long range order. The structural units that constitute the crystal may be atoms, molecules or ions. Crystalline solid material show definitive melting points, and displays sharp characteristic crystalline peaks in an X-ray powder diffractogram (XRPD pattern).

The wording "substantially amorphous" or "substantially XRPD amorphous" as used herein, is defined as picropodophyllin which is physically and chemically stable, which is substantially free from crystalline picropodophyllin, and which continues to exist in a substantially amorphous form under storage conditions such as at a temperature of 20-25 °C and at a relative humidity of 60 %, or at a temperature of 40°C and 75 % relative humidity, for at least 6 months or up to at least one year. During storage under these conditions, there is no presence of crystals as detected by XRPD.

The wording "variability in drug exposure" as used herein, means a variation in plasma concentration upon administration of a certain dose. For example, poor drug exposure may mean that the administered dose might have to increase in order to achieve a clinical effect, but it could also mean that even for an increased dose there may not be any clinical efficacy due to poor uptake of the drug in a particular subject, or group of subjects.

The wording "stable picropodophyllin" as used herein, means amorphous picropodophyllin which is physically stable as well as chemically stable under storage conditions such as at room temperature (20-25°C) and 60 % relative humidity for at least 6 months, or for 1 year or for 2 years under said conditions.

The wording "physically stable picropodophyllin" as used herein, means picropodophyllin which comprises substantially only small amounts of crystalline picropodophyllin, if any.

The wording "increased, or sufficient, solubility of picropodophyllin in aqueous media" is herein defined as picropodophyllin which has a solubility in aqueous media in the mg/ml magnitude.

The wording "sufficient bioavailability" means that picropodophyllin has such a solubility so that it may provide a bioavailability to such an extent that the drug may exert its clinically therapeutic effect when administered to a patient. Unless stabilized, picropodophyllin in solution will form by-products by isomerization or hydrolysis. From a regulatory perspective, it is important that picropodophyllin does not produce by-products spontaneously. The wording "XRPD" or "XRPD pattern" or "XRPD curve" as used throughout the present specification and claims, means an X-ray powder diffractogram, and is used herein to identify the physical properties of picropodophyllin.

During normal storage conditions such as at room temperature (20-25°C) and a relative humidity of 60%, picropodophyllin spontaneously transforms into two unwanted by-products. One of these by-products is podophyllotoxin (PPT) which is highly toxic, and which is the main cytotoxic ingredient of podophyi!in, a resin used for many years for topical treatment of warts. The other by-product of picropodophyllin is the compound podophyllic acid (PA) below.

The wording "chemically stable picropodophyllin" as used throughout the present specification, means that picropodophyllin, shall not spontaneously produce the unwanted by-product podophyllotoxin (PPT) or the by-product podophyllic acid (PA).

The wording "% by wt" as used herein means percentage of weight based on the total weight.

The wording "final pharmaceutical formulation" as used herein, means solid, stabilized

picropodophyllin which is substantially XRPD amorphous, as disclosed and claimed in the present application, formulated into a drug product which is suitable for administration to the subject being treated.

The wording "BRT (below reporting threshhold)" as used herein, means that the amount of each of the biproducts podophyllotoxin or podophyllic acid is below 0.05 % if present in a drug product according to the present invention. A BRT below 0.05 % means that a drug product as disclosed and claimed herein, is chemically stable.

The wording "FDA approved GRAS polymers" as used herein, means a water soluble polymer which is considered safe (Generally recognized as safe) by regulatory authorities such as the FDA.

MEDICAL USE

Solid, stabilized, substantially XRPD amorphous picropodophyllin according to the present invention, may be useful for the treatment of IGF-1R dependent diseases such as cancer.

Examples of cancer indications where picropodophyllin as herein described and claimed, or a pharmaceutical formulation comprising said picropodophyllin, may be useful, are lung cancer such as non-small cell lung cancer (NSCLC) or small cell lung cancer; breast cancer; head and neck cancer such as oral, sinusoidal or pharyngeal cancer; gastrointestinal cancer such as oesophageal cancer, stomach cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor, liver cancer or pancreatic cancer; genitourinary cancer such as prostate cancer, bladder cancer or kidney cancer; gynecologic cancer such as ovarian cancer, cervical cancer, endometric cancer or uterine sarcoma; hematologic cancer such as myeloid leukemia, lymphocytic leukemia, lymphomas or multiple myeloma; musculoskeletal cancer such as Ewings sarcoma, osteosarcoma or soft tissue sarcoma; skin cancer such as malignant melanoma, basal cell cancer, squamous cell cancer or Kaposi's sarcoma; brain and neurologic cancer such as gliomas, glioblastoma, astrocytoma, medulloblastoma, craniopharyngeoma or neuroblastoma; endocrine cancer such as adrenocortical cancer, paraganglioma, pheochromocytoma or thyroid cancer; or eye cancer such as retinoblastoma or uveal melanoma.

One aspect of the present invention, is picropodophyllin as herein described and claimed, or a pharmaceutical formulation comprising said picropodophyllin, for use in the treatment of non-small cell lung cancer (NSCLC) such as adenocarcinoma, squameous or large-cell carcinoma.

Yet an aspect of the present invention is picropodophyllin as herein described and claimed, or a pharmaceutical formulation comprising said picropodophyllin, for use in the treatment of brain and neurologic cancer such as gliomas, glioblastoma, astrocytoma, medulloblastoma,

craniopharyngeoma or neuroblastoma.

Yet an aspect of the present invention is picropodophyllin as herein described and claimed, or a pharmaceutical formulation comprising said picropodophyllin, for use in the treatment of psoriasis; restenosis after coronary angioplasty; diabetes mellitus type 2; nephropathy; eye diseases such as retinopathy or macular degeneration; rheumatoid arthritis; inflammatory bowel disease such as Crohns disease or ulcerative colitis; multiple sclerosis; Alzheimers disease; or graft rejection.

One aspect of the present invention, is a method for the treatment of any medical condition as described herein, whereby picropodophyllin as herein described and claimed, or a pharmaceutical formulation comprising said picropodphyllin, is administered to a patient in need of such treatment.

In one aspect of the invention, there is provided a pharmaceutical formulation comprising picropodophyllin as herein described and claimed, in admixture with a pharmaceutically acceptable adjuvant, diluent and/or carrier.

In one aspect of the present invention, picropodophyllin as herein described and claimed, may be useful in combination with an anti-cancer drug.

In yet an aspect of the invention, picropodophyllin as herein described and claimed, may be administered separately and sequentially, or simultaneously with an anti-cancer agent.

Picropodophyllin as herein described and claimed, may be administered simultaneously with an anti cancer agent as a fix combination such as a pharmaceutical formulation comprising both active drugs in admixture. In yet an embodiment, picropodophyllin as herein described and claimed, may be administered as a fix combination but with each active drug separate from each other.

In yet an embodiment, picropodophyllin as herein described and claimed, may be administered sequentially, i.e. each active drug is administered prior to, or after, the other.

Examples of anti-cancer drugs useful in combination with a compound of formula I as herein described, are cytostatics; targeted anticancer agents being monoclonal antibodies or selective small-molecule inhibitors; hormones; antihormones; or immunostimulating agents.

Examples of cytostatics useful in combination therapy with picropodophyllin as herein described and claimed, are alkylating agents such as melphalan; antimetabolites such as methotrexate or gemcitabine; mitotic inhibitors such as taxanes or vinca alkaloids; cytotoxic antibiotics such as doxorubicin; topoisomerase II inhibitors such as etoposide; or other cytostatics such as cisplatin or carboplatin.

Examples of monoclonal antibodies useful in combination therapy as herein described and claimed, are those targeting the epidermal growth factor receptor (EGFR), HER2, and vascular endothelial growth factor such as trastozumab or bevacizumab.

Examples of selective small-molecule inhibitors useful in combination therapy as herein described and claimed, are those targeting epidermal growth factor receptor, histone deacetylase (HDAC), Raf, platelet-derived growth factor receptors, vascular endothelial growth factor receptor, or c-Kit, such as gefitinib and imatinib.

Examples of hormones useful in combination therapy as herein described and claimed, are estrogens or gestagens.

Examples of antihormones useful in combination therapy as herein described and claimed, are antiestrogens, antiandrogens or enzyme inhibitors.

Examples of immunostimulating agents useful in combination therapy as herein described and claimed, are interferons. In yet an aspect of the present invention, picropodophyllin as herein described and claimed, may be used in combination with radiation therapy, such as external or internal radiation.

In one embodiment of the invention, the external radiation therapy may be selected from external beam radiation therapy (EBRT), three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), image-guided radiation therapy (IGRT), and intraoperative radiation therapy (IORT).

When internal radiation therapy, or brachytherapy, is used in combination with the present invention, radioactive material (seeds or implants) is placed into, or near, the cancer or delivered as an isotope into a vein.

PHARMACEUTICAL FORMULATIONS AND DOSING

In one aspect of the invention, solid, stabilized, substantially XRPD amorphous picropodophyllin as herein described and claimed, may be formulated for oral administration.

In yet an aspect of the invention, solid, stabilized, substantially XRPD amorphous picropodophyllin as herein described and claimed, may be filled into a capsule, compressed into a tablet or a pill, or administered as a solution.

Examples of pharmaceutically acceptable excipients, carriers and/or diluents useful when formulating a drug product as herein described and claimed, are thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, carrier substances, lubricants or binders. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch); fillers (e.g., lactose, glucose, sucrose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, and sodium starch glycolate); wetting agents; diluents; coloring agents; emulsifying agents; pH buffering agents; preservatives; and mixtures thereof.

An aspect of the present invention is a pharmaceutical formulation, wherein solid, stabilized picropodophyllin as herein described and claimed, is filled into a capsule such as hard gelatin capsules or soft gelatin capsules. An aspect of the present invention is a pharmaceutical formulation, wherein solid, stabilized picropodophyllin as herein described and claimed, is in the form of a tablet.

In yet an aspect of the invention, a pharmaceutical formulation as herein described and claimed, may be administered rectally, for example as a suppositorium.

In one aspect of the invention, the dose of solid, stabilized picropodophyllin in the final

pharmaceutical formulation is from 1 mg to 100 mg.

In yet an aspect of the invention, the dose of solid, stabilized picropodophyllin in the final pharmaceutical formulation is from 5 mg to 100 mg.

In one aspect of the invention, the dose of solid, stabilized picropodophyllin in the final

pharmaceutical formulation is from 10 mg to 100 mg.

In one aspect of the invention, the dose of solid, stabilized picropodophyllin in the final

pharmaceutical formulation is from 25 mg to 100 mg.

In one aspect of the invention, the dose of solid, stabilized picropodophyllin as herein described and claimed, in the final pharmaceutical formulation is from 50 mg to 100 mg.

In one aspect of the invention, the dose of solid, stabilized picropodophyllin as herein described and claimed, in the final pharmaceutical formulation is from 10 mg to 50 mg.

In yet an aspect of the invention, the dose of solid, stabilized picropodophyllin as herein described and claimed, in the final pharmaceutical formulation is from 25 mg to 50 mg.

A further aspect of the invention is a pharmaceutical formulation, wherein the daily dosage of picropodophyllin as herein described and claimed, is from 10 mg/day to 500 mg/day.

A further aspect of the invention is a pharmaceutical formulation, wherein the daily dosage of picropodophyllin as herein described and claimed, is from 25 mg/day to 500 mg/day.

A further aspect of the invention is a pharmaceutical formulation, wherein the daily dosage of picropodophyllin as herein described and claimed, is from 50 mg/day to 500 mg/day. An aspect of the invention is a pharmaceutical formulation, wherein the daily dosage of picropodophyllin as herein described and claimed, is from 100 mg/day to 125 mg/day.

In one aspect of the invention, a pharmaceutical formulation comprising picropodophyllin as herein described and claimed, is administered once daily.

In one aspect of the invention, a pharmaceutical formulation comprising picropodophyllin as herein described and claimed, is administered twice daily.

MATERIALS AND METHODS OF PREPARATION

Yet an aspect of the invention is a process for the manufacture of solid, stabilized picropodophyllin as herein described and claimed, said picropodophyllin being substantially XRPD amorphous, wherein:

(i) picropodophyllin is dissolved in at least one organic solvent, providing a solution of picropodophyllin;

(ii) a water-soluble polymer is dissolved in at least one organic solvent, providing a solution thereof;

(iii) the solution of picropodophyllin of step (i) and the dissolved water-soluble polymer of step (ii) are mixed; whereafter

(iv) any remaining solvent is removed by e.g. evaporation, providing solid stabilized

picropodophyllin which is substantially XRPD amorphous.

The starting material Picropodohyllin may be used in any compound form such as amorphous picropodophyllin (non-stabilized), in its free base form, in acid or neutral form or in salt form, as a solvate, or in any crystalline or polymorph form of picropodophyllin.

Examples of organic solvents useful for dissolving picropodophyllin in step (i) may be selected from any one of ethyl acetate, acetone, methanol, ethanol, isopropyl alcohol (IPA), dichloromethane, and diethyl ether.

The organic solvent useful for dissolving the at least one water-soluble polymer in step (ii) may be selected from any one of ethyl acetate, acetone, methanol, isopropyl alcohol (IPA), dichloromethane, and diethyl ether.

The following equipment as shown in Table 1 below, was used during the experiments: Table 1

Picropodophyllin monohydrate used as starting materials in the Examples below, was produced by Galenica AB, Sweden.

EXAMPLES

Hydroxypropyl methylcellulose (HPMC) from Colorcon Asia Pvt Ltd. (trademark Methocel ^® E4M) was used in Example 1 below. Hydroxypropyl methylcellulose (HPMC) from Colorcon Asia Pvt Ltd.

(trademark Methocel ^® E15) was used in Examples 2, 3, 4, 7 and 8A below. Polyvinylpyrrolidone K30 (PVP K30) purchased from Balaji Amines Ltd. was used in Examples 5, 6, and 8B.

Example 1

The effect on the amorphicity of picropodophyllin was studied in this example.

Picropodophyllin monohydrate 2.5 g

Ethyl acetate 450 ml

Methocel ^® E4M (HPMC) 5 g

Ethyl acetate 25 ml

Dichloromethane (DCM) 75 ml

The water-soluble polymer Hydroxypropylmethyl cellulose (HPMC) and picropodophyllin monohydrate was used in a 2:1 ratio.

Amorphous picropodophyllin was prepared as follows:

a) 2.5 g picropodophyllin monohydrate was dissolved in 450 ml ethyl acetate under stirring; b) 5 g Methocel E4M (HPMC) was dissolved in a mixture of 25 ml ethyl acetate and 75 ml dicholoromethane;

c) The solution obtained in step a) was mixed with the solution obtained in step b); and d) Solvent was evaporated from the solution obtained in step c), providing amorphous picropodophyllin as a dry powder, and as confirmed by the XRPD shown in Figure 2.

Example 2

The effect on the amorphicity of picropodophyllin was studied in this example.

Picropodophyllin monohydrate 7.5 g

Acetone 150 ml

Methocel ^® E15 (HPMC) 7.5 g

Acetone 100 ml

Dichloromethane (DCM) 50 ml

The water-soluble polymer Hydroxypropylmethyl cellulose (HPMC) and picropodophyllin monohydrate was used in a 1:1 ratio.

Amorphous picropodophyllin was prepared by following the same procedure as for Example 1. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figure 3.

Example 3

The effect on the amorphicity of picropodophyllin was studied in this example.

Picropodophyllin monohydrate 5 g

Acetone 100 ml

Methocel ^® E15 (HPMC) 2 g

Acetone 30 ml

Dichloromethane (DCM) 45 ml

The water-soluble polymer Hydroxypropylmethyl cellulose (HPMC) and picropodophyllin monohydrate was used in a 1:2.5 ratio. Amorphous picropodophyllin was prepared by following the same procedure as for Example 1. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figure 4.

Example 4

The effect on the amorphicity of picropodophyllin was studied in this example.

Picropodophyllin monohydrate 30 g

Acetone 600 ml

Methocel ^® E15 (HPMC) 30 g

Isopropyl alcohol (IPA) 150 ml

Dichloromethane (DCM) 200 ml

The water-soluble polymer Hydroxypropylmethyl cellulose (HPMC) and picropodophyllin monohydrate was used in a 1:1 ratio.

Amorphous picropodophyllin was prepared by following the same procedure as for Example 1. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figure 5.

Example 5

The effect on the amorphicity of picropodophyllin was studied in this example.

Picropodophyllin monohydrate 2.5 g

Ethyl acetate 450 ml

PVP K30 (Polyvinylpyrrolidone) 7.5 g

Dichloromethane (DCM) 75 ml

The water-soluble polymer polyvinyl pyrrolidone and picropodophyllin monohydrate was used in a 3:1 ratio.

Amorphous picropodophyllin was prepared by following the same procedure as for Example 1. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figure 6.

Example 6

The effect on the amorphicity of picropodophyllin was studied in this example. Picropodophyllin monohydrate 2-5 g

Acetone 50 ml

PVP K30 (Polyvinylpyrrolidone) 7-5 g

Acetone 40 ml

Isopropyl alcohol (IPA) 10 ml

The water-soluble polymer polyvinyl pyrrolidone and picropodophyllin monohydrate was used in a 3:1 ratio.

Amorphous picropodophyllin was prepared by following the same procedure as for Example 1. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figure 7.

Example 7

The effect on the amorphicity of picropodophyllin was studied in this example.

Picropodophyllin monohydrate 5 g

Acetone 100 ml

PVP K30 (Polyvinylpyrrolidone) 5 g

Methocel ^® E15 (HPMC) 5 g

Acetone 150 ml

Isopropyl alcohol (IPA) 20 ml

Dichloromethane 100 ml a) The water-soluble polymers comprised a mixture of polyvinyl pyrrolidone and HPMC in a total amount of 10 g. The ratio water-soluble polymers and picropodophyllin monohydrate was 2:1. b) 5 g picropodophyllin monohydrate was dissolved in 100 ml acetone under stirring;

c) 5 g PVP K30 was dissolved in a mixture of 100 ml acetone and 20 ml isopropyl alcohol;

d) 5 g methocel ^® E15 was dissolved in a mixture of 50 ml Acetone and 75 ml dichloromethane; e) The solution obtained in step b) was mixed with the solution obtained in step a);

f) The solution obtained in step c) was mixed with solution obtained in step d) under stirring; g) Solvent was evaporated from the mixture obtained from step e) until 50% of starting volume remained;

h) 25 ml dichloromethane was added to the mixture obtained in step f);

i) Solvent was evaporated from the mixture obtained from step g), providing amorphous

picropodophyllin as a dry powder, as confirmed by the XRPD shown in Figure 8. Example 8A (Comparative Example)

The effect on the amorphicity of picropodophyllin was studied in this example, where physical mixing of each ingredient was used.

Picropodophyllin monohydrate 2.5 g

Methocel ^® E15 (HPMC) 7.5 g

The water-soluble polymer Methocel ^® E15 (HPMC) and picropodophyllin monohydrate was used in a

3:1 ratio. 2.5 g picropodophyllin monohydrate and 7.5 g Methocel E15 were accurately weighed, providing a physical mixture which was thereafter filled into a capsule.

Example 8B (Comparative Example)

The effect on the amorphicity of picropodophyllin was studied in this example, where physical mixing of each ingredient was used.

Picropodophyllin monohydrate 2.5 g

PVP K30 (Polyvinylpyrrolidone) 7.5 g

The water-soluble polymer PVP K30 (Polyvinylpyrrolidone) and picropodophyllin monohydrate was used in a 3:1 ratio. 2.5 g picropodophyllin monohydrate and 7.5 g PVP K30 (Polyvinylpyrrolidone) were accurately weighed, providing a physical mixture which was filled into a capsule.

STABILITY STUDIES

Example 9 (Comparative Example)

Chemical stability of picropodophyllin in aqueous solution was tested.

Picropodophyllin monohydrate was dissolved in water, saline, PBS and ethanol, respectively, at a concentration of 4 pg/ml. The solutions were kept at room temperature (20 °C) up to 9 months, and analysis of picropodophyllin and by-products were performed by high-performance liquid chromatography (HPLC).

The amount of intact picropodophyllin remaining after storage at 20 °C during 1 month, 3 months and 9 months was measured. Table 2

¹ Saline = 0.154 M aqueous sodium chloride solution; ² PBS = 0.01 M phosphate buffered saline (0.138 M aq. NaCI solution), pH: 7.4; ³ ND = Not determined

From this data, it can be concluded that picropodophyllin is chemically unstable in aqueous solutions and in ethanol, resulting in a significant loss of compound upon storage.

PHARMACEUTICAL FORMULATIONS (DRUG PRODUCTS) AND STABILITY TESTING

A pharmaceutical formulation (Drug Product) is prepared by mixing solid, stabilized, picropodophyllin as herein disclosed and claimed, with pharmaceutically acceptable excipients, carriers, diluents, disintegrants lubricants and/or fillers, providing a pharmaceutical formulation. The formulation may optionally be filled into a capsule. The capsule size selected for a drug product depends on the dose of picropodophyllin to be administered, where capsule size 0 is the largest capsule and capsule size 3 is the smallest capsule.

Example 10

A drug product comprising 25 mg and 50 mg respectively, of picropodophyllin was prepared as follows. Picropodophyllin prepared in accordance with Example 6 (the ratio between

picropodophyllin and water-soluble polymer was 1:3) was mixed with the following ingredients:

Table 3

EXAMPLE 11A-11C

I. Preparation of amorphous picropodophyllin (amorphous API)

Picropodophyllin monohydrate was dissolved in a round bottom flask in acetone, and

polyvinylpyrrolidone (PVP K30 purchased from BASF) was dissolved in a mixture of acetone and isopropyl alcohol (IPA) in a beaker. Both solutions were mixed together, providing a homogeneous solution. The obtained solution was dried using a rotary evaporator, until a solid residue was obtained. Solid material was further scrapped from the round bottom flask and subjected to further drying at 60°C for 10 hours.

The water-soluble polymer polyvinyl pyrrolidone and picropodophyllin monohydrate was used in the ratios shown in Table 4 below. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figures 10 A, B and C.

Table 4

II. Preparation of formulated Drug Product

Amorphous picropodophyllin (amorphous API) of Examples 11A, 11B and 11C respectively, were milled in a mortar pastel (if required) and sieved through an ASTM standard sieve #30 (nominal diameter 600 pm), providing a fine powder which was dried in a hot air oven at 60 °C until loss of drying was below 2%.

The pharmaceutical formulation comprising the excipients as shown in Table 5 below were filled in capsules of size "1", using a manual capsule filling machine. The dose of amorphous API used in each capsule was 50 mg, and the total fill weight per capsule was 230 mg. Amorphous picropodophyllin as formulated into a pharmaceutical formulation (Drug Product) is indicated with DP11A, DP11B and DP11C respectively.

Filled capsules were further packed into a High Density Polethylene Container (HDPE container) together with a desiccant of silica gel.

Table 5

III. Stability testing of formulated Drug Product

The capsules comprising amorphous picropodophyllin of Example 11A, 11B and 11C, were subjected to stability studies for 6 months at 40°C/ 75% RH (Relative Humidity). The stability studies included both chemical stability as well as physical stability.

Results of chemical stability sample analysis are presented in Table 6 below. N.D. means "not detected". BRT means "below reporting threshhold", which herein is 0.05 % .

Table 6

As shown in Figures 13A, B and C respectively, the Drug Products of Example 11A (DP11A), Example 11B (DP11B) and Example 11C (DP11C), were physically stable after storage at 40°C and 75% relative humidity for 3 months.

As shown in Table 6 above, DP11A as well as DP11B did not form the biproducts podophyllotoxin or podophyllic acid during 6 months storage, whereas DP11C formed a minor amount of the biproduct podophyllotoxin which however was below 0.05%. These three drug products are thus chemically stable.

EXAMPLE 12A-12B

I. Preparation of amorphous picropodophyllin (amorphous API)

Picropodophyllin monohydrate was dissolved in a round bottom flask in acetone, and hydroxypropyl methylcellulose (HPMC) (Methocel™ E15 purchased from Colorcon Asia Pvt Ltd) was dissolved in a mixture of acetone and dichloromethane in a beaker. Both solutions were mixed, providing a homogeneous solution. The obtained solution was dried using a rotary evaporator, until a solid residue was obtained. Solid material was further scrapped from the round bottom flask and subjected to further drying at 60°C for 10 hours.

The water-soluble polymer hydroxypropylmethyl cellulose (HPMC E15) and picropodophyllin monohydrate was used in the ratios shown in Table 7 below. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figures 11A and B.

Table 7

II. Preparation of formulated Drug Product

Amorphous picropodophyllin (amorphous API) of Examples 12A and 12B respectively, were formulated into a drug product by following the same procedure as for Examples 11A, B and C above. The ingredients, are shown in Table 8 below and filled in capsules of size "1". The dose of amorphous API used in each capsule was 50 mg, and the total fill weight per capsule was 230 mg. Amorphous picropodophyllin as formulated is indicated with DP 12A and DP 12B respectively.

Table 8

III. Stability testing of formulated Drug Product

The capsules comprising amorphous picropodophyllin of Example 12A and 12B (DP12A and DP12B ), were subjected to stability studies for 6 months at 40°C/ 75% RH (Relative Humidity). The stability studies included both chemical stability as well as physical stability.

Results of chemical stability sample analysis are presented in Table 9 below. N.D. means "not detected". BRT means "below reporting threshhold", which herein is 0.05 % .

Table 9

As shown in Figures 14A and 14 B respectively, the Drug Products of Example 12A (DP12A) and Example 12B (DP12B), were physically stable after storage at 40°C and 75% relative humidity for 3 months.

As shown in Table 9 above, DP12A as well as DP12B did not form the biproducts podophyllotoxin or podophyllic acid during 6 months storage. Both drug products are thus chemically stable. EXAMPLE 13A-13B

I. Preparation of amorphous picropodophyllin (amorphous API)

Picropodophyllin monohydrate was dissolved in a round bottom flask in acetone. Polyvinyl pyrrolidone (PVP K30 purchased from BASF) was dissolved in a mixture of acetone and isopropyl alcohol (IPA) in a beaker. Hydroxypropyl methylcellulose (HPMC) (Methocel™ E15 purchased from Colorcon Asia Pvt Ltd) was dissolved in a mixture of acetone and dichloromethane. Both polymer solutions were added to the picropodophyllin monohydrate solution and mixed, providing a homogeneous solution. The obtained solution was dried using a rotary evaporator until a solid residue was obtained. Solid material was further scrapped from the round bottom flask and subjected to further drying at 60°C for 10 hours.

The water-soluble polymer mixture of polyvinyl pyrrolidone (PVP K30) and hydroxypropylmethyl cellulose (HPMC), and picropodophyllin monohydrate, was used in the ratios shown in Table 10 below. The resulting final dry powder was confirmed as XRPD amorphous picropodophyllin as shown in Figure 12.

Table 10

II. Preparation of formulated Drug Product

Amorphous picropodophyllin (amorphous API) of Examples 13A and 13B respectively, were formulated into a drug product by following the same procedure as for Examples 11A, B and C above. The drug product of Example 13A was filled in capsules of size "1" (DP13A) and the drug product of Example 13B (DP13B) was filled in capsules of size "0". The amount of amorphous API used in each capsule was 50 mg, and the total fill weight per capsule was 230 mg for DP13A and 410 mg for DP13B. Amorphous picropodophyllin as formulated according to Table 11 below is indicated with DP 13A and DP13B respectively.

Table 11

III. Stability testing of formulated Drug Product

The capsules comprising amorphous picropodophyllin of Example 13A (DP13A) and Example 13B (DP13B) were subjected to stability studies for 6 months at 40°C/ 75% RH (Relative Humidity). The stability studies included both chemical stability as well as physical stability.

Results of chemical stability sample analysis are presented in Table 12 below. N.D. means "not detected". BRT means "below reporting threshhold", which herein is 0.05 % .

Table 12

As shown in Figures 15A and 15 B respectively, the Drug Products of Example 13A (DP13A) and Example 13B (DP13B), were physically stable after storage at 40°C and 75% relative humidity for 3 months.

As shown in Table 12 above, DP13A as well as DP13B did not form the biproducts podophyllotoxin or podophyllic acid during 6 months storage. Both drug products are thus chemically stable. DISSOLUTION STUDIES

Example 14

In-vitro dissolution profiles of picropodophyllin according to Example 4, Example 5, and comparative Example 8A and Example 8B, was measured in 0.1% SLS at 37°C in 900ml dissolution media at 100 rpm using a USP Type II dissolution apparatus. Picropodophyllin prepared according to each of Example 4, Example 5, Example 8A and Example 8B, was formulated into a drug product according to Example 10 above, and filled into a capsule of size 0.

Table 13

The in-vitro dissolution data show that picropodophyllin prepared according to the present invention (Example 4 and Example 5) provides an improved dissolution rate compared to the dissolution rate for picropodophyllin which has been prepared as a physical mixture (Example 8A and Example 8B). The numerical values are expressed as percentages of dissolved active ingredient in relation to starting quantities.

Table 13 shows the amount of picropodophyllin as drug product (i.e. picrpodophyllin according to the invention, filled into a capsule) which is dissolved after 15 minutes, 30 minutes, 45 minutes and 60 minutes, respectively.

Example 15

In-vitro dissolution profiles of picropodophyllin according to Examples 11A, 11B and 11 C were performed at 100 rpm using a USP Type II dissolution apparatus. 6.8 g sodium

dihydrogenphosphate monohydrate was dissolved in 1000 ml water, pH was adjusted to 6.8 with sodium hydroxide (NaOH) and 5 g sodium lauryl sulfate (SLS) was added and mixed properly.

The bath temperature was 37 °C ±0.5°C and 900 ml dissolution media was used.

Dissolution was measured at 15, 30, 45 and 60 minutes for each drug product at start of stability testing, and after storage during 1 month, 3 months and 6 months. Table 14 A

Table 14 B

Table 14A and 14 B shows the amount of picropodophyllin released after 15 minutes, 30 minutes,

45 minutes and 60 minutes, respectively.

Example 16

In-vitro dissolution profiles of picropodophyllin according to Examples 12A and 12B were performed by following the procedure of Example 15.

Table 15

Dissolution was measured at 15, 30, 45 and 60 minutes for each drug product at start of stability testing, and after storage during 1 month, 3 months and 6 months.

Table 15 shows the amount of picropodophyllin released after 15 minutes, 30 minutes, 45 minutes and 60 minutes, respectively.

Example 17

In-vitro dissolution profiles of picropodophyllin according to Examples 13A and 13B were performed by following the procedure of Example 15.

Table 16

Dissolution was measured at 15, 30, 45 and 60 minutes for each drug product at start of stability testing, and after storage during 1 month, 3 months and 6 months.

BIOLOGICAL TESTING

Example 18

Pharmacokinetic (PK) study

Picropodophyllin prepared according to the present invention was administered orally to each individual group of Male Wistar rats (n=6) corresponding to a dose of 75 mg picropodophyllin/kg body weight. By diluting picropodophyllin according to Example 2, Example 6 and Example 7 respectively, with phosphate buffer containing 0.2% Tween 80 (Polysorbate 80) at a concentration of 25 mg picropodophyllin/ml homogeneously by shaking manually for 20 minutes.

A dose of 3mg/kg body weight was administered to each Male Wistar rats.

The oral suspension as used in the phase I clinical trial as disclosed in Ekman S et al; Acta Oncologica, 2016; 55: pp. 140-148, was administered to each Male Wistar rat as control in this experiment. Plasma sample was collected retro-orbital sinus node at 0 min, 15 min, 30 min, 1 hr, 1.5 hr, 2 hr, 4 hr, 6 hr, 8 hr and 24 hr. Pharmacokinetic parameters were evaluated including Tmax, Cmax, Tl/2, AUC, which are summarized in the following Table 5 below.

The pharmacokinetic data given in Table 5 below, show that picropodophyllin according to the present invention provides an improved bioavailability compared to the oral suspension used in the Phase I clinical trial (Simon et al., J Clin Oncol 30, 2012 (suppl; abstr 7539). Table 17

*Oral suspension used in Applicant ^' s Phase I clinical trial (Simon et al., J Clin Oncol 30, 2012

(suppl; abstr 7539))

Picropodophyllin according to the present invention provides an improved dissolution rate of the therapeutic compound picropodophyllin, and this improvement of dissolution rate is reflected in the improvement of bioavailability. Example 19

Pharmacokinetic (PK) study from Phase I study

Pharmacokinetic data from 3 female patients treated with increasing doses of picropodophyllin formulated as an oral suspension. Plasma concentrations of picropodophyllin were measured following repetitive sampling during a 8-12 hour period after the dose had been ingested in the morning. Based on the plasma concentrations the area under the curve (AUC, here expressed as pM*h) was then calculated.

Table 18

¹ NT = Not tested. As shown in Table 18 above, the bioavailability (AUC) showed a large and significant variability both within the patients, as well as between patients and there was no, or only a poor, positive correlation with the doses given.

Example 20

Assessment of pharmacokinetic (PK) profile of picropodophyllin

A clinical Phase I SAD (single ascending dose) trial is being performed in patients with solid tumors. The number of patients to participate in the study is approximately 24 and the study is being performed in India. A drug product of Example 10A is used in this study.

Male/female patients above the age of 18 years with Histologic or cytologic diagnosis of a solid tumor is participating in the study. Patients must have advanced disease and deemed refractory to standard anticancer therapy or are not expected to benefit or prolong survival from any

standard anti-cancer therapy.

Single dose (once daily administration) under fasted conditions will be performed at all dose levels. Single dose under both fasted and fed conditions with 7 days follow-up (a total of 7 days after each single dose, with day of dosing considered as day 1) will be performed at 25 mg and 50 mg dose.

In addition, single-dose PK analysis under both fasting and fed conditions will be performed in additional cohorts with doses defined as MTD (Maximum Tolerated Dose) or dose levels below MTD. A multiple dose study will be initiated only after completion of the single dose study.

Total 13 blood samples will be taken for PK analysis from each patient. Four (4) ml of blood will be collected at pre-dose (before dosing), 0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 3.00, 4.00, 6.00, 8.00 12.00, and 24.00 hours after dose administration. Plasma concentration-time profiles will be analyzed by non compartmental analysis (NCA) to determine Cmax, time to maximum plasma concentration (Tmax), elimination half-life (tl/2), time to last analytically quantifiable concentration (Tz), terminal elimination rate constant (lz), area under the concentration-time curve AUCO-t, AUCO-inf, apparent clearance after oral administration (CL/F), and apparent terminal volume of distribution after oral administration (Vz/F). NCA PK analysis will be performed using Phoenix WinNonlin Version 8.0 or higher Pharsight Corporation, USA. Statistical analyses will be performed by using the SAS ^® package (SAS Institute Inc., USA, version 9.4 or higher). Mean, minimum, maximum, standard deviation, median, mode and coefficient of variation will be calculated for plasma concentration of pharmacokinetic parameters (Cmax, AUCO-t, AUC0- , Tmax and tl/2).