Abstract:
The present invention relates to pharmaceutical compositions comprising a topoisomerase I inhibitor including, but not limited to, a camptothecin derivative.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to pharmaceutical compositions comprising a topoisomerase I inhibitor, including but not limited to a camptothecin derivative. 
       BACKGROUND OF THE INVENTION 
       [0002]    Camptothecin derivatives are a class of compounds described in U.S. Pat. No. 6,242,457. Camptothecin derivatives, such as those disclosed in U.S. Pat. No. 6,242,457, present highly specific difficulties in relation to administration generally, including in particular problems of drug bioavailability because these derivatives have very poor water solubility. 
         [0003]    7-t-Butoxyiminomethylcamptothecin is a quinoline-based alkaloid blocking, through a topoisomerase inhibition, cell division in cells that divide rapidly, such as cancer cells. The drug substance is very poorly soluble in aqueous media which hinders the delivery of the effective amount of drug to the cancer cells. 
         [0004]    In addition, 7-t-butoxyiminomethylcamptothecin is susceptibly to hydrolysis, and at physiological pH (˜7.4) the lactone ring tends to open readily, resulting in drug inactivation. In blood plasma the lactone ring is quickly opened to create the carboxylate form of the drug, which is poorly accumulated in cancer cells. Once internalized by the cancer cells, the carboxylate form exhibits no activity against its molecular target, topoisomersase I. Thus, the hydrolysed product is ineffective at treating cancer. Therefore, there is a need to develop a drug formulation comprising camptothecin derivatives, including but not limited to 7-t-butoxyiminomethylcamptothecin, that is stable, able to deliver clinical relevant dose to the cancer cells and is easy to use. 
       SUMMARY OF THE INVENTION 
       [0005]    The present invention overcomes the instability and poor solubility problems of camptothecin derivatives, including 7-t-butoxyiminomethylcamptothecin, when administered in its free form, by forming an unique pharmaceutically active composition containing functionalized phospholipids. This stabilized formulation can be used for an iv and subcutaneous administration. 
         [0006]    In another aspect, the invention is directed:
       a) to stabilize and increase the circulating time of a camptothecin derivative, including, but not limited to, 7-t-butoxyiminomethylcamptothecin, in blood; and   b) to increase the drug anti-tumor efficacy and to improve the action on a wider range of cancer diseases, through a drug targeting strategy.       
 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0009]    The active agent is an inhibitor of topoisomerase I (Topo I inhibitor) and is therefore capable of preventing disease symptoms that are caused inter alia by the activation of the topoisomerase I receptor. 
         [0010]    The camptothecin derivatives of the present invention, which are described in U.S. Pat. No. 6,242,457 include:
   7-methyoxyiminomethylcamptothecin;   7-methoxyiminomethyl-10-hydroxycamptothecin;   7-(tert-butoxycarbonyl-2-propoxy)iminomethylcamptothecin;   7-ethoxyiminomethylcamptothecin;   7-isopropoxyiminomethylcamptothecin;   7-(2-methylbutoxy)iminomethylcamptothecin;   7-t-butoxyiminomethylcamptothecin;   7-t-butoxyiminomethyl-10-hydroxycamptothecin;   7-t-butoxyiminomethyl-10-methoxycamptothecin;   7-(4-hydroxybutoxy)iminomethylcamptothecin;   7-triphenylmethoxyiminomethylcamptothecin;   7-carboxymethoxyiminomethylcamptothecin;   7-(2-amino)ethoxyiminomethylcamptothecin;   7-(2-N,N-dimethylamino)ethoxyiminomethylcamptothecin;   7-allyloxyiminomethylcamptothecin;   7-cyclohexyloxyiminoethylcamptothecin;   7-cyclohexylmethoxyiminomethylcamptothecin;   7-cyclooctyloxyiminomethylcamptothecin;   7-cyclooctylmethoxyiminomethylcamptothecin;   7-benzyloxyiminomethylcamptothecin;   7-[(1-benzyloxyimino)-2-phenylethyl] camptothecin;   7-(1-benzyloxyimino)ethylcamptothecin;   7-phenoxyiminomethylcamptothecin;   7-(1-t-butoxyimino)ethylcamptothecin;   7-p-nitrobenzyloxyiminomethylcamptothecin;   7-p-methylbenzyloxyiminomethylcamptothecin;   7-pentafluorobenzyloxyiminomethylcamptothecin;   7-p-phenylbenzyloxyiminomethylcamptothecin;   7-[2-(2,4-difluorophenyl)ethoxy]iminomethylcamptothecin;   7-(4-t-butylbenzyloxy)iminomethylcamptothecin;   7-(1-adamantyloxy)iminomethylcamptothecin;   7-(1-adamantylmethoxy)iminomethylcamptothecin;   7-(2-naphthyloxy)iminomethylcamptothecin;   7-(9-anthrylmethoxy)iminomethylcamptothecin;   7-oxiranylmethoxyiminomethylcamptothecin;   7-(6-uracyl)methoxyiminomethylcamptothecin;   7-[2-(1-urcyl)ethoxy]iminomethylcamptothecin;   7-(4-pyridyl)methoxyiminomethylcamptothecin;   7-(2-thienyl)methoxyiminomethylcamptothecin;   7-[(N-methyl)-4-piperidinyl]methoxyiminomethylcamptothecin;   7-[2-(4-morpholininyl]ethoxy]iminomethylcamptothecin;   7-(benzoyloxyiminomethyl) camptothecin;   7-[(1-hydroxyimino)-2-phenylethyl) camptothecin;   7-tert-butyloxyiminomethylcamptothecin-N-oxide; and   7-methoxyiminomethylcamptothecin N-oxide.   
 
         [0056]    In a very preferred embodiment of the invention, the topoisomerase I inhibitor of formula (I) has the following structure known as Compound A: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0057]    The preferred and especially preferred active agents, in free or pharmaceutically acceptable salt form, may be prepared as described in U.S. Pat. No. 6,424,457. As mentioned therein, they may be in the form of their possible enantiomers, diastereoisomers and relative mixtures, the pharmaceutically acceptable salts thereof and their active metabolites. 
         [0058]    In one embodiment the present invention provides a pharmaceutical composition comprising:
       a) a therapeutically effective amount of 7-t-butoxyiminomethylcamptothecin entrapped in liposomes; and   b) a pharmaceutically acceptable excipient.       
 
         [0061]    The present invention provides a stable, highly pharmacologically active formulation by solubilizing the drug in phospholipids comprising 7-t-butoxyiminomethylcamptothecin. The formulation is in the form of liposomes, comprised of multiple phospholipids, such as conventional phospholipid, such as phosphatidylcholine cholesterol and the functionalized lipid. Typically, 7-t-butoxyiminomethylcamptothecin binds the lipid bilayer the membrane of liposome with high affinity. The 7-t-butoxyiminomethylcamptothecin intercalates between the acyl chains of the lipid, thereby reducing the lactone ring of the drug from interacting with the aqueous environment inside and outside the liposomes and thus protected from hydrolysis. 
         [0062]    The liposome composition of the present invention is composed primarily of vesicle-forming lipids. Such a vesicle-forming lipid is one which:
       a) can form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids, or   b) is stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its head group moiety oriented toward the exterior and interior, polar surface of the vesicle.       
 
         [0065]    The vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or non-polar. There are a variety of synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and sphingomyelin, where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. The above-described lipids and phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods. Other suitable lipids include glycolipids and sterols such as cholesterol or cholesterol derivatives. 
         [0066]    Preferred diacyl-chain lipids for use in the present invention include diacyl glycerol, such as phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), sphingomyelin (SPM) and the like, alone or in combination. 
         [0067]    The present invention overcomes the instability and poor solubility problems of 7-t-butoxyiminomethylcamptothecin when administered in its free form by forming an unique pharmaceutically active composition containing functionalized phospholipids. The functionalized phospholipids are those that surface grafted with certain hydrophilic polymers, and/or with certain ligands. 
         [0068]    The surface drafted hydrophilic polymer is formed by including, at least in the outer lipid layer of the liposomes. Suitable hydrophilic polymers that are intended to extend liposome-circulation time, include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol and polyaspartamide. 
         [0069]    In a preferred embodiment, the hydrophilic polymer is polyethyleneglycol, preferably as a PEG chain having a molecular weight between 500-10,000 daltons, typically between 1,000-5,000 daltons. 
         [0070]    The surface grafted liposome provided by the hydrophilic polymer chains provides colloidal stability and serves to protect the liposomes from uptake by the reticulo-endothelial system, providing an extended blood circulation lifetime for the liposomes to reach the target cells. The extent of enhancement of blood circulation time is preferably several fold over that achieved in the absence of the polymer coating. 
         [0071]    Examples of specific ligands for liposomes functionalization may include folic acid, peptides, proteins, enzymes, lectins, biotin, avidin, mono-, oligo-, and polysaccharides, hormones, cytokines, polyclonal and monoclonal antibodies including chimeric and humanized ones and their fragments. 
         [0072]    In another embodiment the present invention provides a method of treating a cellular proliferative disease comprising administering to a mammalian host a pharmaceutical composition comprising:
       a) a therapeutically effective amount of 7-t-butoxyiminomethylcamptothecin entrapped in liposomes; and   b) a pharmaceutically acceptable excipient
 
The mammalian host may be a human.
       
 
         [0075]    In a further embodiment the present invention provides the use of a pharmaceutical composition of the present invention for the treatment of disease symptoms that are caused by the activation of the topoisomerase I receptor. 
         [0076]    In a further embodiment the present invention provides use of the composition of the present invention for the preparation of a medicament for the treatment of disease symptoms that are caused by the activation of the topoisomerase I receptor. 
         [0077]    The stabilized 7-t-butoxyiminomethylcamptothecin formulation circulates for prolonged period with better drug retention and plasma stability, leading to either passive or active preferential location into the tumor cells (as compared to a conventional formulation) through the Enhanced Permeation and Retention (EPR) effect and/or targeted delivery through specific cell surface receptors recognition. The stabilized 7-t-butoxyiminomethylcamptothecin formulation can be used for an iv and subcutaneous administration. 
         [0078]    In a human of about 70 kg body weight, for example, from about 0.5-5 mg 7-t-butoxyiminomethylcamptothecin per kg of body weight can be administered. Preferably, about 1.0-3.0 mg of 7-t-butoxyiminomethylcamptothecin per kg of body weight is administered. However, it can be necessary to deviate from the dosages mentioned and in particular to do so as a function of the nature and body weight of the subject to be treated, the nature and the severity of the illness, the nature of the preparation and if the administration of the medicine, and the time or interval over which the administration takes place. Thus, it can suffice in some cases to manage with less that the above-mentioned amount of active compound whilst in other cases the above-mentioned amount of active compound must be exceeded. The particular required optimum dosage and the type of administration of 7-t-butoxyiminomethylcamptothecin can be determined by one skilled in the art, by available methods. Suitable amounts are therapeutically effective amounts that do not have excessive toxicity, as determined in empirical studies. 
         [0079]    With the pharmaceutical compositions of the present invention, 7-t-butoxyiminomethylcamptothecin could be safely and effectively delivered by intravenous administration or into other body compartments. 
         [0080]    The benefits of the present invention are that we could solve the problem of 7-t-butoxyiminomethylcamptothecin poor solubility and the low stability of the molecule in physiological pH intended to be used for iv and/or subcutaneous administration. Additional benefits are that with liposomes grafted with certain polymers we could increase the circulation time through the EPR effect and, through a functionalization of the liposomes/micelles with specific ligands, we could transport and enhance the cell internalization of 7-t-butoxyiminomethylcamptothecin to the targeted tumor cells more effectively compared to a conventional formulation. 
         [0081]    7-t-Butoxyiminomethylcamptothecin can also be stabilized by entrapping in the hydrophobic region of micelles, and bound to the micelle membrane. 
         [0082]    The present invention is directed:
       a) to stabilize and increase the circulating time of the 7-t-butoxyiminomethylcamptothecin in blood; and   b) to increase the drug anti-tumor efficacy and to improve the action on a wider range of cancer diseases, through a drug targeting strategy.       
 
       EXAMPLES 
     Example 1 
     7-t-Butoxyiminomethylcamptothecin in Small Unilamellar, Long-Circulating Liposomes: Surface Grafted with Certain Polymers (Ex. Peg: Polyethylene Glycol). 
     7-t-Butoxyiminomethylcamptothecin in PEG-Liposome 
       [0085]    The sample was prepared following the thin film hydration method also called Bangham method (Ref. Bangham A. D. &amp; al., J. Mol. Biol. 13, 238-252, 1965) with the following adaptations: 
         [0000]    STEP 1: preparation of the drug substance (DS), lipid film. Excipients and DS are dissolved in Ethanol. The organic solvent is evaporated off on a rotavapor (Rotavap R-210/215 from Büchi Switzerlans) for 4 hr at 40° C. to obtain a very homogenous DS, lipid film. The thin film obtained is maintained on rotavap for 2 hr, 55° C. and 30 mbar.
 
STEP 2: hydration of the DS, lipid film. To the DS, lipid film is added PB-Man buffer solution (pH 7.4) under magnetic stirring and at 40° C. for 30 min. A milky solution is obtained: the liposomal solution. The solution is put in an ultra-sound bath for 10 min at RT.
 
STEP 3: Freeze thawing of the liposomal solution. The liposomal solution is put in a liquid nitrogen (until solidification) and warmed in a 40° C. water bath (until melting) for 3 cycles.
 
STEP 4: Extrusion of the liposomal solution. The liposomal solution is extruded (LIPEX® Extruder from Norther Lipids Inc.) through polycarbonate filters (400 and 100 nm).
 
STEP 5: Sterilization. The liposomal solution is filtered on a sterile Millipore® filter 0.2 μm.
 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Sample composition 
               
             
          
           
               
                   
                 Concentration 
                   
               
               
                 Ingredients 
                 (mg/mL) 
                 Volume (mL) 
               
               
                   
               
             
          
           
               
                 Phosphatidylcholine 
                 50 
                 — 
               
               
                 MPEG-2000-DSPE 
                 13 
                 — 
               
               
                 Cholesterol 
                 5 
                 — 
               
               
                 D,L α-tocopherol 
                 0.25 
                 — 
               
               
                 7-t-Butoxyiminomethylcamptothecin 
                 0.15 
                 — 
               
               
                 Phosphate buffer pH 5.4 
                 — 
                 30 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
             
               
               
               
             
               
               
             
               
               
               
             
               
               
             
           
               
                   
               
               
                 Analytical characterization 
               
             
          
           
               
                 Analytical test 
                 Results 
               
               
                   
               
             
          
           
               
                 Appearance (by visual examination) 
                 Slightly green-yellow, 
               
               
                   
                 translucent dispersion 
               
               
                 pH-value 
                 5.44 
               
               
                 HPLC identification of the DS 
                 Positive 
               
             
          
           
               
                 HPLC assay 
                 0.11 
                 mg/mL 
               
             
          
           
               
                 HPLC degradation products 
                 Each ≦ 0.5%; sum ≦ 2.0% 
               
             
          
           
               
                 Mean particle size 
                 102 
                 nm 
               
               
                 Particle size distribution 
                 99% &lt; 282 
                 nm 
               
               
                 Specific turbidity 
                 527.1 
                 NTU 
               
             
          
           
               
                 Drug encapsulation 
                 96% 
               
               
                   
               
             
          
         
       
     
       Example 2 
     7-t-Butoxyiminomethylcamptothecin in Liposomes with Ligands: Surface Grafted with Certain Ligands for a Drug Targeting Strategy 
     7-t-Butoxyiminomethylcamptothecin in PEG-Liposome 
       [0086]    The sample was prepared following the thin film hydration method as described in example 1. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Sample composition 
               
             
          
           
               
                   
                 Concentration 
                   
               
               
                 Ingredients 
                 (mg/mL) 
                 Volume (mL) 
               
               
                   
               
             
          
           
               
                 Phosphatidylcholine 
                 100 
                 — 
               
               
                 MPEG-2000-DSPE 
                 26 
                 — 
               
               
                 Cholesterol 
                 10 
                 — 
               
               
                 D,L α-tocopherol 
                 0.5 
                 — 
               
               
                 7-t-Butoxyiminomethylcamptothecin 
                 0.25 
                 — 
               
               
                 Phosphate buffer pH 5.4 
                 — 
                 10 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Analytical characterization 
               
             
          
           
               
                   
                 Analytical test 
                 Results 
               
               
                   
                   
               
               
                   
                 Appearance (by visual examination) 
                 Slightly green-yellow, 
               
               
                   
                   
                 translucent dispersion 
               
             
          
           
               
                   
                 Mean particle size 
                 122 
                 nm 
               
               
                   
                 Particle size distribution 
                 99% &lt; 402 
                 nm 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Stability test at 5° C. and 25° C. 
               
             
          
           
               
                   
                 Mean particle size 
                 Mean particle size 
               
               
                 Time (weeks) 
                 (nm) at 5° C. 
                 (nm) at 25° C. 
               
               
                   
               
               
                 0 
                 122 
                 122 
               
               
                 1 
                 125 
                 124 
               
               
                 2 
                 127 
                 128 
               
               
                 4 
                 127 
                 127 
               
               
                 8 
                 124 
                 126 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Plasma stability test at 37° C. 
               
             
          
           
               
                   
                 Mean particle size 
                 Mean particle size 
               
               
                 Time (hrs) 
                 (nm) in 50% plasma 
                 (nm) in 70% plasma 
               
               
                   
               
             
          
           
               
                 0 
                 129 
                 131 
               
               
                 0.75 
                 123 
                 121 
               
               
                 1.5 
                 120 
                 118 
               
               
                 3 
                 119 
                 118 
               
               
                 6 
                 120 
                 119 
               
               
                 24 
                 126 
                 121 
               
               
                   
               
             
          
         
       
     
       Example 3 
     7-t-Butoxyiminomethylcamptothecin in Long-Circulating Phospholipids Micelles: Surface Grafted with Certain Polymers (Ex. PEG2000: Polyethylene Glycol) 
     7-t-Butoxyiminomethylcamptothecin in PEG-Liposome 
       [0087]    The sample was prepared following the thin film hydration method as described in the example 1. The only difference is in the STEP 4, the extrusion of the liposomal solution through polycarbonate filters (100 and 50 nm). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Sample composition 
               
             
          
           
               
                   
                 Concentration 
                   
               
               
                 Ingredients 
                 (mg/mL) 
                 Volume (mL) 
               
               
                   
               
             
          
           
               
                 Phosphatidylcholine 
                 100 
                 — 
               
               
                 MPEG-2000-DSPE 
                 26 
                 — 
               
               
                 Cholesterol 
                 10 
                 — 
               
               
                 D,L α-tocopherol 
                 0.5 
                 — 
               
               
                 7-t-Butoxyiminomethylcamptothecin 
                 0.25 
                 — 
               
               
                 Phosphate buffer pH 5.4 
                 — 
                 40 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Analytical characterization 
               
             
          
           
               
                   
                 Analytical test 
                 Results 
               
               
                   
                   
               
               
                   
                 Appearance (by visual examination) 
                 Slightly green-yellow, 
               
               
                   
                   
                 translucent dispersion 
               
               
                   
                 pH-value 
                 5.40 
               
             
          
           
               
                   
                 Mean particle size 
                 108 
                 nm 
               
               
                   
                 Particle size distribution 
                 99% &lt; 293 
                 nm 
               
               
                   
                 Osmolarity 
                 362 
                 mOsm 
               
               
                   
                 Specific turbidity 
                 538.9 
                 NTU 
               
               
                   
                 Residual ethanol 
                 &lt;0.98% 
                 (m/m) 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Stability test at 5° C. and 25° C. 
               
             
          
           
               
                   
                 Mean particle size 
                 Mean particle size 
               
               
                 Time (weeks) 
                 (nm) at 5° C. 
                 (nm) at 25° C. 
               
               
                   
               
               
                 0 
                 108 
                 108 
               
               
                 1 
                 105 
                 106 
               
               
                 2 
                 103 
                 103 
               
               
                 4 
                 104 
                 104 
               
               
                   
               
             
          
         
       
     
       Example 4 
     7-t-Butoxyiminomethylcamptothecin in Long-Circulating Phospholipids Micelles: Surface Grafted with Certain Polymers (Ex. PEG2000: Polyethylene Glycol) and with Certain Ligands (e.g., Folic Acid) 
     7-t-Butoxyiminomethylcamptothecin in Folic Acid Functionalized PEG-Liposome 
       [0088]    The sample was prepared following the thin film hydration method as described in the example 1. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Sample composition 
               
             
          
           
               
                   
                 Concentration 
                   
               
               
                 Ingredients 
                 (mg/mL) 
                 Volume (mL) 
               
               
                   
               
             
          
           
               
                 Phosphatidylcholine 
                 50 
                 — 
               
               
                 MPEG-2000-DSPE 
                 12.5 
                 — 
               
               
                 Folic acid funct. PEG-DSPE 
                 0.5 
                 — 
               
               
                 Cholesterol 
                 5 
                 — 
               
               
                 D,L α-tocopherol 
                 0.25 
                 — 
               
               
                 7-t-Butoxyiminomethylcamptothecin 
                 0.15 
                 — 
               
               
                 Phosphate buffer pH 5.4 
                 — 
                 10