Polymer-active ingredient conjugate

Abstract

The invention relates to a polymer-active ingredient conjugate and a process for producing the polymer-active ingredient conjugate. The polymer-active ingredient conjugate comprises an active ingredient selected from aryl amino compounds with conjugateable functional groups; and a water soluble polymer, wherein the active ingredient is attached to the water soluble polymer by a covalent bond that is cleavable by biological or physico-chemical means.

Description

[0001] POLYMER-ACTIVE INGREDIENT CONJUGATE

[0002] BACKGROUND TO THE INVENTION

[0003] THIS invention relates to a polymer-active ingredient conjugate, in particular a water-soluble polymer-active ingredient conjugate and more particularly to a process for producing the polymer-active ingredient conjugate, in particular the water-soluble polymer-active ingredient conjugate.

[0004] For over a decade now the WHO has dissuaded the use of monotherapies for treatment in P. falciparum endemic countries and has strongly recommended the artemisinin combination therapy (ACT) (World Health Organization, 2015). This regimen mainly consists of a fast-acting artemisinin-based drug taken in combination with a longer-acting drug which often is an aryl alcohol compound like lumefantrine [Lumf] (World Health Organization, 2015)(Ramharter et al., 2008)(Koram et al., 2005). This combination treatment has had a significant positive impact on malaria mortality and morbidity. However, the gains are at risk of being reversed as treatment failures and drug resistant strains of P. falciparum are beginning to emerge. Artemisinin resistance is now prevalent in many far-eastern Asian countries. Re-admission of patients for severe malaria after ACT treatment has been reported in some countries (Siddiqui et al., 2015). Severe malaria is treated with parenterally administered artesunate for at least 24 h followed by the full three days ACT therapy (World Health Organization, 2015)(World Health Organization, 2005)(Li and Weina, 2010). This is primarily because there is no injectable water-soluble form of lumefantrine. This is a highly effective and lifesaving treatment regimen. However, this will require hospitalization for the full 24 h. The second part of the regimen is usually the responsibility of the patient or family members as it is carried out at home. Lessons from other diseases like tuberculosis show that patients tend to show much lower compliance after recovering from the initial severe clinical state. This blindsides the WHO’s recommendation that ACT must not be a monotherapy.

[0005] It is an object of the invention to provide a water-soluble lumefantrine that will, at least partially, alleviate the above disadvantages.

[0006] It is an object of the invention to provide a water-soluble lumefantrine which will be a useful alternative to existing ACT.

[0007] SUMMARY OF THE INVENTION

[0008] According to a first embodiment of the invention there is provided a polymer-active ingredient conjugate, in particular a water-soluble polymer- active ingredient conjugate, comprising:

[0009] • an active ingredient, typically selected from aryl amino compounds such as lumefantrine, desbutyllumefantrine and halofantrine with conjugateable functional groups such as, but not limited to, hydroxyls, amines, aldehydes, thiols; and

[0010] • a water soluble polymer, typically having a molecular weight of below 100 KDa, preferably below 60 KDa and more preferably below 50 KDa, and typically 1 KDa and above, typically selected from biocompatible polyacrylamide (10 KDa to 40 KDa), polyether (1 KDa to 10 KDa) and polyamide (5 KDa to 40 KDa) derivatives with hydroxyl, amino, thiol, aldehyde, carboxylic acid or any other derivatizeable functionalizable groups, polyamides, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, chitosan (low molecular weight, less than 6000 Da), low molecular weight polysaccharides, poloxamers, poly(styrene-maleic acid), poly(dimethylamino)ethyl methacrylate, poly glutamic acid (PGA) and other polyacrylamide derivatives i.e. p-NAM-sfaf-p-AA; wherein the active ingredient is attached to the water soluble polymer by a stable covalent bond that may be cleavable by biological or physico-chemical means such as enzyme, pH, temperature, agitation or radiation, in vivo.

[0011] The covalent bond may be a scissile bond.

[0012] The aryl amino compounds have a low solubility in water of less than 1 x 102mg / ml, typically less than 1 x 103mg / ml, typically less than 2 x 104mg / ml, for example, between 1 x 107mg / ml to 1 x 103mg / ml, preferably between 1 x 106mg / ml to 5 x 103mg / ml and more preferably between 1 x 105to 2 x 104mg / ml.

[0013] The polymer-active ingredient conjugate is preferably between 10 to 106, more preferably 102to 105, more preferably 104times more soluble in water compared to the active ingredient alone.

[0014] The polymer-active ingredient conjugate may be for use in combination with artesunate salts, primaquine or primaquine conjugates, dihydroartemisinin or other artemisinin-based conjugates; preferably in co-administration. The administration is preferably in a single regimen.

[0015] According to a second embodiment of the invention there is provided a process for producing a polymer-active ingredient conjugate, in particular a water-soluble polymer-active ingredient conjugate, comprising the following steps:

[0016] A.1 ) reacting an active ingredient, typically selected from aryl amino compounds such as lumefantrine, desbutyllumefantrine and halofantrine with conjugateable functional groups such as, but not limited to, hydroxyls, amines, aldehydes, thiols, with succinic anhydride, succinic acid, amino acids, hydroxyl acids, thiol acids and any organic molecule with a chemical functional group that could be activated to react with the hydroxyl or conjugateable group on the active ingredient; and

[0017] A.2) attaching a water soluble polymer, typically having a molecular weight of below 100 KDa, preferably below 60 KDa and more preferably below 50 KDa, and typically 1 KDa and above, typically selected from biocompatible polyether (1 KDa to 10 KDa) derivates with hydroxyl, amino, thiol, aldehyde, carboxylic acid or any other derivatizeable functional groups, poloxamers, chitosan (low molecular weight, less than 6000 Da), and any other low molecular weight polysaccharides, N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers, and polyamides; to the active ingredient product produced in step A.1 ) to produce a polymer-active ingredient conjugate, in particular a water-soluble polymer-active ingredient conjugate; or

[0018] B) attaching a water soluble polymer, typically having a molecular weight of below 40 KDa, preferably below 10 KDa and more preferably below 5 KDa, typically selected from poly glutamic acid (PGA), poly(dimethylamino)ethyl methacrylate, poly(styrene-maleic acid), and biocompatible polyamides (5 KDa to 40 KDa) and polyacrylamide (10 KDa to 40 KDa) derivatives i.e. p- NAM-sfaf-p-AA with hydroxyl, amino, thiol, aldehyde, carboxylic acid or any other derivatizeable functionalizable groups, directly to the active ingredient to produce a polymer-active ingredient conjugate without step a);

[0019] wherein steps A) or B) occur in the presence of a suitable solvent and base catalyst, or acyl transfer agent.

[0020] Trimethylamine may be further added to the reaction in step A).

[0021] The solvent is typically selected from anhydrous dimethylformamide (DMF), anhydrous dichloromethane (DCM), anhydrous acetonitrile, anhydrous pyridine, anhydrous ethyl acetate. The base catalyst is preferably 4-(dimethylamino)-pyridine, pyridine, imidazole, alkanamines such as methylamine, triethylamine, N, N- diisopropylethylamine, guanidine, histidine.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows a 1 H Nuclear Magnetic Resonance (NMR) spectrum of lumefantrine (Lumf) (600 MHz, CDC ).

[0024] Figure 2 shows a 1 H NMR spectrum of lumefantrine-succinic acid

[0025] (Lumf-Suc) conjugates (600 MHz, CDCb)

[0026] Figure 3 shows a 1 H NMR spectrum of (Polyethylene qivcoS) PEG

[0027] (600 MHz, D2O)

[0028] Figure 4 shows a 1 H NMR spectrum of Polyethylene glycol-succinic acid lumefantrine (PEG-Suc-Lumf) conjugate (600 MHz, CDCb).

[0029] Figure 5 shows a 1 H NMR spectrum of p-NAM-stat-p-AA-Lumf conjugate (600 MHz, CDCb).

[0030] Figure 6 is a graph of size distribution by intensity of polyethylene glycol-lumefantrine conjugate (A); and p-NAM-sfaf-p-AA- lumefantrine conjugate (B).

[0031] Figure 7 is an illustration of lumefantrine-succinic acid conjugate in a yellow powder form.

[0032] DESCRIPTION OF AN EMBODIMENT OF THE INVENTION

[0033] Lumefantrine (or benflumetol) is a synthetic drug used to treat, in combination with artemether, acute uncomplicated malaria or cerebral malaria. This combination is also administered after initial treatment of complicated malaria with artesunate, a water-soluble artemisinin derivative. The option of co-administering lumefantrine with artesunate as an intravenously injected combination therapy has not been available because lumefantrine is completely insoluble in water. The reported solubility is 3.09 x 105mg / ml. Its reported logP is between 8.34 and 9.19. We have chemically synthesized a water soluble form of lumefantrine by reversibly linking the drug to a water soluble polymer.

[0034] The present invention relates to lumefantrine and other related drugs which belong to another class of drugs, the aryl amino alcohols. These drugs are used as combination drugs only in malaria.

[0035] The solubility of the active ingredients in water may vary for example less than 1 x 102mg / ml, typically less than 1 x 103mg / ml, typically less than 2 x 104mg / ml, for example, between 1 x 107mg / ml to 1 x 103mg / ml, preferably between 1 x 106mg / ml to 5 x 103mg / ml and more preferably between 1 x 105to 2 x 104mg / ml. The solubility of the active ingredients in water may also be in the range of 1 .1 1 x 104mg / ml to 3.091 1 x 105.

[0036] We have, by applying the polymer therapeutics techniques, synthesized a polymer-drug conjugate of lumefantrine which has increased the solubility of the drug more than 103times. This has been done without permanently altering the drug. The drug is linked via a stable covalent bond that may be cleaved by biological or physicochemical means, typically a scissile chemical bond, to the hydrophilic polymer. Under specific physiological conditions the unaltered original drug is released. This fact means that the therapeutic potential is not altered through the conjugation or solubilizing process. Any free lumfantrine was completely washed off from the conjugate using different purification techniques. Selection of an appropriate water-soluble polymer (e.g. polyethylene glycol, biocompatible polyacrylamide, polyether and polyamide derivative with hydroxyl, amino, thiol, aldehyde, carboxylic acid or any other functionalizable groups, poly glutamic acid, N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers, chitosan (low molecular weight, less than 6000 Da), poloxamers, poly(styrene-maleic acid), poly(dimethylamino)ethyl methacrylate and other polyacrylamide derivatives i.e. p-NAM-sfaf-p-AA) to make the drug soluble. The molecular weight for polyether derivatives may vary for example from 1 KDa to 10 KDa, Polyamide derivatives may vary for example from (eg PGA) 5 KDa to 40 KDa and Polyacrylamide derivatives i.e. p-NAM-stat-p-AA may vary for example from 10 KDa to 40 KDa. Conjugation or functionalizing with small molecules like sulphates does not make lumefantrine soluble. Succinic acid, the molecule conjugated to artemisinin to make it water-soluble does not make lumefantrine soluble. Not all water-soluble polymers will make lumefantrine soluble. Low molecular weight polymers are used in the present invention that provides for a water-soluble conjugate system for lumefantrine and other aryl amino alcohols of its class. The molecular weight of the polymers are for example below 100 KDa, preferably below 60 KDa and more preferably below 50 KDa, and typically 1 KDa and above

[0037] The polymer-active ingredient conjugate is a water-soluble formulation which may be used for direct intravenous injection.

[0038] Conditions for successful reaction. We explored several reaction conditions before successfully discovering the appropriate conditions for conjugation to occur.

[0039] To a stirred solution of acid; Lum-Succ (0.063 g, 0.10 mmol) in anhydrous DMF (5 ml) was added the amine; PEG-NH2 (0.400 mg, 0.08 mmol), EDAC (0.094 g, 0.60 mmol) and Sulfo-NHS (0.046 g, 0.40 mmol). The reaction was allowed to proceed at 40 °C under a condenser for 48 h. The reaction was terminated when TLC showed complete consumption of the starting acid and appearance of a UV active spot on the baseline.

[0040] To a stirred solution of acid; p-NAM-stat-p-AA (0.200 g, 0.015 mmol) in anhydrous DMF (5.0 ml) was added the alcohol Lumf (0.1 18 g, 0.224 mmol), ED AC (0.009 g, 0.045 mmol) and Sulfo-NHS (0.0097 g, 0.045 mmol). The reaction was allowed to proceed at 40 °C under a condenser for 48 hours. The reaction was terminated when TLC showed complete consumption of the starting acid and appearance of a UV active spot on the baseline.

[0041] The key benefit of this technology is that it will, for the first time, provide the opportunity for an intravenous artemisinin combination therapy (ACT). It opens the opportunity for severe malaria to be treated with a single regimen in the clinic without a follow-up full oral dose. This will have the advantage of actually reducing the cost of treating severe malaria which has a high mortality rate, especially among children.

[0042] Example 1 : Polyethylene Glvcol-Lumefantrine Synthesis Reaction

[0043] The general synthetic route to accomplish PEG-o-Succ-Lumf and PEG-NH- Suc-Lumf conjugate is shown in Scheme 2 and Scheme 3; respectively. In all reactions the coupling to the Lumf was achieved using EDAC / sulfo-NHS with succinic acid as a linker.

[0044] Two reactions were carried out,

[0045] a) Lumefantrine was reacted with succinic anhydride and a drop of trimethylamine (TEA) was added to the reaction (Scheme 1 ),

[0046] Scheme 1 : Synthesis of Lumf-Suc conjugate. and

[0047] b) The free carboxylic acid group on a newly formed prodrug of lumefantrine was used to attach to PEG-OH and PEG-NH2.

[0048]

[0049]

[0050] Scheme 2: Synthesis route of PEG-o-Suc-Lumf (n = 5000 Da).

[0051] Cl

[0052]

[0053] Scheme 3: Synthesis route of PEG-NH-Suc-Lumf (n = 5000 Da).

[0054] In all reactions above, anhydrous DMF and 4-(dimethylamino)-pyridine (DMAP) were used as solvent and base catalyst or acyl transfer agent; respectively.

[0055] Example 2: The p-NAM-sfaf-p-AA-Lumefantrine Synthesis Reaction The polymer, p-NAM-sfaf-p-AA, polyacrylamide derivative was used to conjugate lumefantrine directly using EDAC / sulfo-NHS through the lumefantrine hydroxyl group.

[0056]

[0057] EDAC / sulfo-NHS,

[0058] anhydrous DMF, 40 °C

[0059] n=90 m=10 reflux,

[0060] 48 h

[0061] OH

[0062]

[0063] Scheme 4: Synthesis route of p-NAM-stat-p-AA-Lumf conjugate.

[0064] With reference to Figures 1 -7, the NMR spectrum of pure lumefantrine (Figure 1 ) and lumefantrine-succinic acid conjugates (Figure 2) in CDCI3. The successful O-succinylation of the alcohol on the lumefantrine resulted in the downfield shift of the chiral proton to 5.73-5.67 ppm from 5.37-5.34 ppm.1FI NMR of the Polyethylene glycol-succinic acid-lumefantrine (PEG- Succ-Lumf) conjugate depicted corresponding peaks of both Lumf and PEG (Figure 4). The typical methylene protons of the PEG backbone were observed at 3.61 ppm and characteristic proton peaks (CH) of the aromatic group of Lumf were between 7.276-7.689 ppm. The proton NMR of p-NAM- sfaf-p-AA-lumefantrine conjugate followed the same array. The polymer- Lumf conjugates have amphiphilic characteristics due the hydrophilicity of the polymeric material used and the hydrophobic nature of the drug. Average particle size below 200 nm was obtained for PEG-Succ-Lumf conjugate irrespective of the type of linkage (Figure 6). The average particle size of p-NAM-sfaf-p-AA-lumefantrine conjugate was below 100 nm (Figure 6). Despite the choice of the polymer, the poly dispersity index (PDI) values were always below 0.2, which is an indication of the relatively homogeneous size distribution achieved with carbodiimide chemistry.

[0065] REFERENCES

[0066] Koram, K.A., Abuaku, B., Duah, N., Quashie, N., 2005. Comparative efficacy of antimalarial drugs including ACTs in the treatment of uncomplicated malaria among children under 5 years in Ghana. Acta Trop. 95, 194-203. https: / / doi.Org / 10.1016 / j.actatropica.2005.06.018 Li, Q., Weina, P., 2010. Artesunate: The best drug in the treatment of severe and complicated malaria. Pharmaceuticals 3, 2322-2332. https: / / doi.org / 10.3390 / ph3072322

[0067] MALARIA CONTROL TODAY, 2005.

[0068] Ramharter, M., Kurth, F., Schreier, A.C., Nemeth, J., Glasenapp, I. von, Belard, S., Schlie, M., Kammer, J., Koumba, P.K., Cisse, B., Mordmuller, B., Lell, B., Issifou, S., Oeuvray, C., Fleckenstein, L., Kremsner, P.G., 2008. Fixed-Dose Pyronaridine-Artesunate

[0069] Combination for Treatment of Uncomplicated Falciparum Malaria in Pediatric Patients in Gabon. J. Infect. Dis. 198, 91 1-919. https: / / doi.Org / 10.1086 / 591096

[0070] Siddiqui, M.R., Willis, A., Bil, K., Singh, J., Mukomena Sompwe, E., Ariti, C., 2015. Adherence to Artemisinin Combination Therapy for the treatment of uncomplicated malaria in the Democratic Republic of the Congo. F1000Research 4, 1-16. https: / / doi.Org / 10.12688 / f1000research.6122.2

[0071] World Health Organization, 2015. Treatment of Severe Malaria. Guidel.

[0072] Treat. Malar. 71-88. https: / / doi.Org / 10.1016 / 0035-9203(91 )90261 -V

Claims

CLAIMS1 . A polymer-active ingredient conjugate, comprising:• an active ingredient selected from aryl amino compounds with conjugateable functional groups; and• a water soluble polymer;wherein the active ingredient is attached to the water soluble polymer by a covalent bond that is cleavable by biological or physico-chemical means.

2. The polymer-active ingredient conjugate claimed in claim 1 , wherein the covalent bond is cleavable by biological or physico-chemical means includes enzyme, pH, temperature, agitation or radiation.

3. The polymer-active ingredient conjugate claimed in any of the preceding claims, wherein the covalent bond is a scissile bond.

4. The polymer-active ingredient conjugate claimed in any one of the preceding claims, wherein the aryl amino compounds have a solubility in water of less than 1 x 102mg / ml.

5. The polymer-active ingredient conjugate claimed in claim 4, wherein the aryl amino compounds have a solubility in water of less than 1 x 103mg / ml.

6. The polymer-active ingredient conjugate claimed in claim 5, wherein the aryl amino compounds have a solubility in water of less than 2 x 10-4mg / ml.

7. The polymer-active ingredient conjugate claimed in any one of the preceding claims, wherein the conjugateable functional groups are selected from hydroxyls, amines, aldehydes, and thiols.

8. The polymer-active ingredient conjugate claimed in any one of the preceding claims, wherein the aryl amino compound is selected from lumefantrine, desbutyllumefantrine and halofantrine.

9. The polymer-active ingredient conjugate claimed in any one of the preceding claims, wherein the water soluble polymer has a molecular weight of below 100 KDa.

10. The polymer-active ingredient conjugate claimed in claim 9, wherein the water soluble polymer has a molecular weight of below 60 KDa.1 1. The polymer-active ingredient conjugate claimed in claim 10, wherein the water soluble polymer has a molecular weight of below 50 KDa.

12. The polymer-active ingredient conjugate claimed in any one of the preceding claims, wherein the water soluble polymer is selected from biocompatible polyacrylamide, polyether and polyamide derivatives with hydroxyl, amino, thiol, aldehyde, carboxylic acid or any other functionalizable groups, polyamides, N-(2- hydroxypropyljmethacrylamide (HPMA) copolymers, chitosan, polysaccharides, poloxamers, poly(styrene-maleic acid), poly(dimethylamino)ethyl methacrylate and other polyacrylamide derivatives.

13. The polymer-active ingredient conjugate claimed in any one of the preceding claims, which is between 10 to 106times more soluble in water compared to the active ingredient alone.

14. The polymer-active ingredient conjugate claimed in claim 13, which is between 101to 105times more soluble in water compared to the active ingredient alone.

15. The polymer-active ingredient conjugate claimed in claim 14, which is between 103to 104times more soluble in water compared to the active ingredient alone.

16. The polymer-active ingredient conjugate as claimed in any one of the preceding claims, for use in combination with artesunate salts, primaquine or primaquine conjugates, dihydroartemisinin or other artemisinin-based conjugates.

17. The polymer-active ingredient conjugate as claimed in any one of the preceding claims, for use in co-administration with artesunate salts, primaquine or primaquine conjugates, dihydroartemisinin or other artemisinin-based conjugates.

18. The polymer-active ingredient conjugate as claimed in claim 17, for administration in a single regimen.

19. A process for producing a polymer-active ingredient conjugate, comprising the following steps:A.1 ) reacting an active ingredient selected from aryl amino compounds with conjugateable functional groups with succinic anhydride, succinic acid, amino acids, hydroxyl acids, thiol acids and any organic molecule with a chemical functional group that may be activated to react with the hydroxyl or conjugateable group on the active ingredient;A.2) attaching a water soluble polymer to the active ingredient product produced in step A.1 ) to produce a polymer-active ingredient conjugate; orB) attaching a water soluble polymer directly to the active ingredient to produce a polymer-active ingredient conjugate; wherein steps A) or B) occur in the presence of a suitable solvent and base catalyst, or acyl transfer agent.

20. The process claimed in claim 19, wherein the aryl amino compounds have a solubility in water of less than 1 x 102mg / ml.

21. The process claimed in claim 20, wherein the aryl amino compounds have a solubility in water of less than 1 x 103mg / ml.

22. The process claimed in claim 21 , wherein the aryl amino compounds have a solubility in water of less than 2 x 104mg / ml.

23. The process claimed in any one of claims 19 to 22, wherein theconjugateable functional groups are selected from hydroxyls, amines, aldehydes, and thiols.

24. The process claimed in any one of claims 19 to 23, wherein the aryl amino compound is selected from lumefantrine, desbutyllumefantrine and halofantrine.

25. The process claimed in any one of claims 19 to 24, wherein the water soluble polymer has a molecular weight of below 100 KDa.

26. The process claimed in claim 25, wherein the water soluble polymer has a molecular weight of below 60 KDa.

27. The process claimed in claim 26, wherein the water soluble polymer has a molecular weight of below 50 KDa.

28. The process claimed in any one of claims 19 to 27, wherein thewater soluble polymer at step A.2) is selected from biocompatible polyether derivates with derivatizeable functional groups,poloxamers, chitosan, and polysaccharides, N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers, andpolyamides.

29. The process claimed in any one of claims 19 to 27, wherein the water soluble polymer at step B) is selected from polyamides, poly glutamic acid (PGA), poly(styrene-maleic acid), and biocompatible polyamides and polyacrylamide derivatives with derivatizeable functionalizable groups.

30. The process claimed in any one of claims 19 to 29, whereintrimethylamine is added to the reaction in step A.1 ).

31. The process claimed in any one of claims 19 to 30, wherein the solvent is selected from anhydrous dimethylformamide (DMF), anhydrous dichloromethane (DCM), anhydrous acetonitrile, anhydrous pyridine, and anhydrous ethyl acetate.

32. The process claimed in any one of claims 19 to 31 , wherein the base catalyst is selected from 4-(dimethylamino)-pyridine, pyridine, imidazole, and alkanamines.

33. The process claimed in claim 32, wherein the base catalyst is selected from methylamine, triethylamine, N, N- diisopropylethylamine, guanidine, and histidine.