PHARMACEUTICAL COMPOSITION CONTAINING A MIXTURE OF PROENZYMES AND ENZYMES
Pharmaceutical composition containing a mixture of proenzymes and enzymes, containing proenzymes trypsinogen and chymotrypsinogen and enzymes ct-amylase and lipase as active substances, and one or more pharmaceutically acceptable excipients, for simultaneous, separate and subsequent administration of the composition in parenteral or transmucosal way, the composition has anti-proliferative and anti-metastatic effects to cancer tumours and is intended for therapeutic, prophylactic and anti-metastatic use in mammals.
The invention deals with new pharmaceutical compositions containing a mixture of proenzymes and enzymes having anti-proliferative and anti-metastatic effects.
BACKGROUND ARTMalignant neoplastic diseases represent a vast group of diseases that are one of the worst curable death causes. They cause 13 percent of deaths per year recently. (Jemal A. et al., CA: Cancer J. Clinic., 61, 2011, 69-90.). Occurrence of malignant tumours brings dangers given by the ability of tumour cells to change adjacent cells while new blood vessels, further supporting cells and metastases are produced.
Anti-neoplastic pharmacotherapy is an important part of large spectrum of present treatment approaches. In terms of therapeutic position pharmacotherapy of neoplastic diseases is divided to adjuvant (affecting so called residual disease e.g. after a surgical operation), non-adjuvant (preceding operation and radiation treatment, aimed at tumour devitalisation and inhibition) and metro therapy (long-term application of minimum doses of cytostatics affecting neoangiogenesis in advanced forms of neoplastic diseases.
Involved authorities like World Health Organisation WHO, Federal Drug Administration (FDA), European Medicines Agency (EMA) or State Institution for Drug Control (SUKL, Prague, CR) register more than 260 substances for oncology disease pharmacotherapy recently, classified by WHO (Collaborating Centre for Drug Statistics Methodology) according to application, therapeutic and chemical criteria into three main ATC subgroups: (http://www.whocc.no/; on line 25. 6. 2012): 1. Cytostatics (with 5 subgroups), 2. Hormonal medicines (2 subgroups), 3. Imunostimulants (4 subgroups) and 4. immunosuppressives (5 subgroups). About 160 substances of immunostimulant and immunosuppresive groups have FDA license at present. With regard to ATC classification numerous details of anti-neoplastic substances and their mechanism activities can be found for example in current monograph Avendano, C., Menendez, J. C.: Medicinal Chemistry of Anticancer Drug. Elsevier, 2008, Amsterodam. 441 s.
Antimetabolites, mitotic inhibitors, hormonal function inhibitors, reactive radical substances, photosensitizers, DNA alkylation agents, DNA separation spindle interactors, intercalators and topoisomerase inhibitors, tubulin and microtubules, attacking substances, inhibitors of cancer growth signals and proliferation are described there in the list of substances and mechanisms.
Classification based on cellular biologic mechanisms seems to be very interesting from theoretical point of view as well as for practical purposes. It fully respects the fact that mutual interaction between drug, tumour and its host is logically responsible for the final response to therapy (Wu, Xi-Z.: Medical Hypotheses 66, 2006, 883-887). From this viewpoint anti-neoplastic drugs are divided to cytotoxic substances and biological cell modifiers that kill tumour cells. They are often used as basic medicines. The other two groups, biological response modulators and biochemical modulators contain substances that are considered medicines used for adjuvant combined therapy.
Results of recent intensive research into mechanisms of effects of cytostatics and immunomodulation anti-neoplastic substances and progress in biopharmaceutical research have brought extremely wide spectre of findings but also new questions.
Now it is clear that normal cell growth, activity and functions are coordinated and regulated by a multilateral network of signal pathways that receive extracellular signal molecules and via a cascade of proteins and activation of gene transcription coordinate a wide scale of processes like growth, proliferation, invasion and apoptosis (Dowanward, J., Nature 411, 2001, 759-762). New strategies are aimed at protein-protein type interactions (Wells, J. A., McClendon, C. L., Nature 450, 2007, 1001-1009), which are undoubtedly also used in our new composition with enhanced role of tertiary structure of substances of this type both on pharmacodynamic parameters and particularly inherently pharmacokinetic parameters.
We can add in general that any intervention in such a complicated and still little known system always leads to some consequences, which are however not always known or predictable or positive. Drawbacks of present pharmacotherapy in oncology are also linked to them.
Disadvantages of Present Oncologic PharmacotherapyHuman carcinomas occur as subsequence of various factors and their cells themselves further influence cellular signalling and paths regulating cellular proliferation and the time of survival of the other cells. Complex cellular signalling in a cancer cell is modified, the number of stimuli that tumours react to decreases, but the reaction intensity increases. This represents the base of increased sensitivity of tumours to genotoxic stresses and immune influences (O'Driscoll, L., Cur. Cancer Drug Targets 9, 2009, 250-251).
Cancer treatment success varies a lot today depending on particular malignity type. Some types of cancer diseases, e.g. testicular seminoma, infant leukaemia, and some lymfoms are very sensitive to anti-neoplastic treatment (Gonzalez-Angulo, A. M et al., Adv. Exp. Med. Biol., 608, 2007, 1-22). Other malignant cancer diseases show a limited response only (if any) and no efficient therapy is available against them now. (Jemal, A. et al., CA Cancer J. Clin., 60, 2010, 277-300). In the instances of advanced tumours with developed metastases, chemotherapy remains palliative treatment in better cases. If we define success rate of present pharmacotherapy by the survival time of cancer patients, we find that this essential parameter of treatment factually has nearly not changed for the last 30 years. Achieved success has actually to be attributed to timely diagnostics (Hemminki, K., Annals Oncol., 23, 2012, 760-764). Moreover, most of the clinically approved anti-neoplastic medicines are characterized by narrow therapeutic window, which is particularly related to their high systemic toxicity (Lowenthal, R. M.; Eaton, K., Hematol. Oncol. Clin. North Am., 10, 1996, 967-90).
Resistance against anti-neoplastic medicines represents another serious problem, particularly in long-term treatment (Redmond, K. M. et al.: Front. Biosci., 13, 2008, 5138-5154), whether based internally in tumour cells (intrinsic resistivity) or it is acquired. Multiple resistance against higher number of anti-neoplastic substances, often of different structures and functions, appears more and more often (Wu, Ch.-P. et al.; Curr. Pharm. Biotechnol., 12, 2011, 609-620). This clinical resistance is multifactorial and heterogeneous with numerous molecular mechanisms. (Glickman, M. S., Sawyers, C., Cell 148, 2012, 1089-98). Relatively short history of targeted biological cancer treatment has already been filled with wide spectrum of resistances (Gorre, M. E. et al., Science 293, 2001, 876-880).
Probably the most critical aspects of oncologic pharmacotherapy are linked to higher risk of unwanted induction of immunogenic responses or toxic effects as most of the newer medicines belonging to groups ATC 3 and ATC 4 are supramolecular active substances mainly of biotechnological origin. They belong to so called biopharmaceuticals, today biologics, protein therapeuticals, and each of them has actually a potential to affect hundreds of physiological processes in a patient (Yang, J. A.; Hastings Sci. Tech. L. J., 3, 2011, 217, 1-18), which represents substantial growth compared to medicines with small molecule. The risks of immune responses include hypersensitiveness, anaphylaxis, pseudoallergic anaphylactoid reaction, series disease, reaction to infusion, therapeutic effect reduction (Borges, S. et al., Clin. Pharmacol. Ther. 74, 2006, 61-74; Barbosa, M.D.F.S., Drug Disc. Today 16, 2011, 345-53), generation of antibodies against the medicine and cross reaction between antibodies for therapeutic and endogenous proteins (Wager K., Jones, G.: Cur. Biotechnol., 297, 2012, 297-317). This certainly brings extended demands for production conditions (Singh, S. K., J. Pharm. Sci., 100, 2011, 354-87), including control mechanisms (EMA. 2007. Doc. Ref. EM EA/CH MP/BMWP/14327/2006.
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/W C500003946.pdf, which affects the price of the medicines. This is also the reason why these medicines are often used not only monotherapeutically, but also in combinations with conventional chemotherapeutics, sequentially and in link to further physical and/or surgical methods.
The fact that in 98 percent of cases it is only linked to parenteral injection or infusion administration is another disadvantage of present pharmacology of tumours by substances of biologics group. This means that parenteral administration approach and related medicine forms are elaborated in detail. On the other hand all principal disadvantages of parenteral administration itself remain unsolved, from higher risk of infection in the needle mark to patient non-compliance even in the link to the hospital and its qualified staff. Vast majority of biologics have only a short plasma half-life and has to be applied by infusion.
The above reference to the existing disadvantages of oncologic pharmacotherapy means in general that alternative conceptual and practical approaches to anti-neoplastic disease treatment are still required (Sachlos, E., et al., Cell 149, 2012, 1284-1297), and that the existing range of anti-neoplastic substances has to be extended by medicines that do not have the above briefly summarized disadvantages or are at least able to reduce them.
DISCLOSURE OF THE INVENTIONThe solution is based on a pharmaceutical composition containing a mixture of proenzymes and enzymes, containing proenzymes trypsinogen and chymotrypsinogen and enzymes α-amylase and lipase as active substances, and one or more pharmaceutically acceptable excipients, for simultaneous, separate and subsequent administration of the composition in parenteral or transmucosal way, while the composition has anti-proliferative and anti-metastatic effects to cancer tumours and is intended for therapeutic, prophylactic and anti-metastatic use in mammals.
The pharmaceutical composition has advantageous ratio of enzymatic active substances, namely activities of trypsinogen (T), chymotrypsinogen A (CH), α-amylase B.s (A) and lipase T.a. (L) for T:CH:A:L ratio expressed in m.u. in the range from 150:150:40:1 to 400:1200:200:1
The above pharmaceutical composition advantageously contains trypsinogen of type I, chymotrypsinogen of type A, α-amylase is produced by Bacillus sp. and lipase is from Triticum aestivum.
The minimum enzymatic activity of active substances in the pharmaceutical composition according to the invention is advantageously as follows: trypsinogen 40 m.u./mg, chymotrypsinogen 60 m.u./mg, α-amylase 20 m.u./mg and lipase 1 m.u./mg.
At least one of the active substances in the pharmaceutical composition according to the invention is advantageously replaced with biologically similar substance obtained by extraction from higher plants, animals or by cultivation procedures using mould cells, yeast cells, or bacteria, the primary structure of the biologically similar substance with the active substance which it has replaced in the composition being at least 70% identical and the position of active places essential for the effect is at least in 95% identical.
The pharmaceutical composition according to the invention is particularly suitable for systemic sublingual, rectal, inhalation or parenteral administration.
The pharmaceutical composition according to the invention may contain numerous substances as pharmaceutically acceptable excipients, particularly one or more hydrophilic polyhydric alcohols including polyethylene glycol with mol. weight 100 to 8000 and/or hydrophilic low molecular alcohols like glycerol, propylene glycol, n-propanol, and/or saccharides like trehalosa, mannitol, lactose, sorbitol, myo-inositol, and/or polysorbates like polysorbate 20, polysorbate 60, polysorbate 80, poloxamers like poloxamer 182, poloxamer 417, poloxamer 908, and/or one or more lipophilic excipients including hydrogenated triglycerides like hydrogenated glycerol trioleate, hydrogenated glycerol-cocoate, and/or esters of higher fatty acids with glycerol or propylene glycol like glycerol-tripalmitate, glycerol-trioleate, glycerol-tristearate, glycerol-distearate, glycerol-dioleate, glycerol-monolaurate, propylene glycol-myristate, glycerol-dipalmitostearate, and/or esters of lower monovalent alcohols like diisopropyl-adipate, isopropyl-laurate, isopropyl-linoleate, isopropyl-palmitate, and/or esters of higher fatty acids with medium and higher fatty alcohols, including myristyl-strearate, capryl-stearate, cetyl palmitate, caprin-behenate, lauroyl oleate, and/or higher fatty alcohols, including lauryl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol, behenyl alcohol and analogously higher fatty acids like lauric, myristic, palmitic, stearic, lignoceric, arachidonic, behenic acids and their ethoxylated derivatives like polyethylene glycol 10 oleyl alcohol, polyethylene glycol 25 stearyl alcohol, polyethylene glycol 40 stearyl alcohol, stearoyl polyethylene glycol 32 glycerol, polyethylene glycol 15 hydroxy stearate, and/or vegetable oils, including cottonseed oil, sunflower oil, groundnut oil, soya oil, castor oil, and their ethoxylated derivatives like polyoxyl 35 ricinoleate, and/or phospholipids including egg lecithin, soya lecithin, dioleoylphosphatidylcholine, dipalmitoylphosphatidylserine, and/or sterols including cholesterol and its derivatives like cholesteryl-linoleate, cholesteryl-acetate, and/or biocompatible and biodegradable polymers particularly polyesters like poly-DL-lactic acid (PDLLA), polyglycolic acid (PGA), poly-DL-lactic glycolic acid (PLGA).
If the pharmaceutical composition is designed for sublingual administration, it is advantageously in the form of nanofibres, while it contains at least one of polyvinyl polymers like polyvinylpyrrolidone with molecular weight approx. 30 to 50 thousand and polyvinyl alcohols with molecular weight from 20 thousand to 200 thousand, of cellulose derivatives like methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose and/or polysaccharides of starch type like hydroxyethyl starch, carboxymethyl starch sodium salt and/or dextrins with molecular weight from 4 000 to 80 000 and/or of biotechnological polysaccharides of dextran type with molecular weight from 10 000 to 80 000, and/or glucuronate type substances like xanthan mucilage, and/or further polyuronides especially their salts, particularly sodium, potassium, like hyaluronans, alginans, pectinans, arabinans and/or polymers based on acrylic, methacrylic acids and/or their copolymers like carboxyvinyl polymers (carbomers) netted with s polyalkenyl ethers of sugars or poly alcohols (like diallyl sucrose a diallylpentaerythritol, biodegradable polyesters of α-hydroxy acids like (PDLLA), (PGA), (PLGA), polycaprolactones with molecule weight from 10 to 100 thousand, further polymeric excipients of copolymer type like polyvinyl caprolactam-polyvinyl acetate polyethylene glycol.
If the pharmaceutical composition or its part is designed for inhalation administration it advantageously also contains at least one or more saccharides, including trehalose, mannitol, glucose and/or various forms of lactose.
The pharmaceutical composition according to any of the claims may advantageously be in the form of nanofibre stabilized preparation for direct administration of active substances or as stabilized storage of active substances in an intermediate product or in the final preparation.
It is a composition of proenzymes and enzymes representing a substantial modification of enzyme therapy by its composition and efficiency. It solves the main side effects of present oncologic treatment based particularly on impact on lively divided healthy tissues like gastrointestinal system mucous membrane, medulla, liver and kidney parenchyma. This is thus targeted biological therapy in the real sense of the word, non-toxic, selectively focused on tumour cells, wide spectral from the point of view of anti-neoplastic effects. It impacts on carcinomas, sarcomas, as well as acute haematological malignity.
Dosage of the therapeutic composition is only limited by the minimum daily dose in relation to the volume of degrading elements originating from tumour cells. Thanks to the intrinsic non-toxicity of the composition according to the invention, where particularly, or only degrading products from decomposed tumour cells may have toxic effect, the composition may also be used for diagnostic purposes and actually with regard to the experience gained from application of in vivo methods of testing the efficiency on lines of tumour cells grown on mice it is obvious that the composition according to this application will have anti-neoplastic effect not only in humans, but its effect against tumours affecting animals, e.g. a dog or a cat may also be anticipated. However their different anatomy as well as immunogenicity has to be taken into account, while such influences can hardly be predicted.
The minimum daily dose means such a quantity of the anti-neoplastic composition that ensures full or partial therapy of a tumour diseases or the required diagnostic or prophylactic effect with regard to the current state of the tumour disease and in relation to the chosen administration method.
Stabilization of the individual components of the anti-neoplastic four-composition according to this application is solved for the purpose of elaboration into a pharmaceutical preparation and for the administration purposes by means of excipients and procedures ensuring preservation and actually regeneration of the secondary and higher supramolecular structures of the partial active components.
Systemic administration in non-invasive ways, which may be advantageously applied to the anti-neoplastic composition according to the invention solves one of the important today's problems of biologic medicine administration (biologics, protein therapeutics etc.), which is the short plasmatic half-time of these substances in organism.
DETAILED DISCLOSURE OF THE INVENTIONThe new solution according to the invention is fundamental, empirically observed from influences on trophoblast as a biologic model of malignant tumours even at the molecular level (Soundarajan, R., Rao, J., Reprod. Biol. Endocrinol., 2, 2004, 15 (http://www.rbej.com/content/2/1/15, on line: 30.6.2013;). The solution is based on classic concept of J. Beard (Beard, J.: Lancet 168, 1905, 281-283), who proposed treatment of advanced carcinomas by fresh pancreatic extracts. Their antitumoural activity was based on proteolytic potential. He assumed that enzymes produced by pancreas limit trophoblastic invasion and he concluded that pancreatic extracts should have similar inhibition effects to invasive tumours. In the following years extract from pancreatic enzymes were thoroughly examined for that time. It was found that they really very efficiently inhibit cancer growth even in patients at advanced stage of malignant neoplasm (Goeth, R. A., J. Am. Med. Assoc. 1907; 1030). With regard to several subsequent reports on negative effects of administration of then very imperfectly processed pancreatic extracts the research was abandoned for a long period (Gurchot, Ch., Oncology 31, 1975, 310-333). Later traceable exceptions from recent years (e.g. Maeda et al.: EU Pat. 0215 662 A2, 1986) only mention proteases, not protease proenzymes, zymogens.
The hypothesis that proenzymes and not activated enzymes are the crucial components of extract effects was first formulated and then elaborated in detail by F. Trnka (Trnka, F. et al.: EU Pat. 0743 070 A2, 1996; CZ Pat. 283 972, 1998; U.S. Pat. No. 5,858,357; 1999). About thirty years ago this author discovered and experimentally confirmed the fact that a mixture of trypsinogen, chymotrypsinogen and amylase, approximately in the concentrations proposed by J. Beard, has strong anti-tumoral, anti-invasive and anti-angiogenic effect. One of the effects of the above proenzymes and their mixture with amylase is inhibition of tumour cells migration at the cellular level. F. Trnka at al. also discovered (Novak, J., Trnka, F.: Anticancer Res., 25, 2005, 1157-77), that continuous exposition of tumour cells to low concentration of the above substances leads to formation of cell aggregates and inhibition of metastases. He managed to combine the historic data with new findings and he deduced that
(a) protease proenzymes are resistant to inactivation of protease inhibitors,
(b) proenzyme activation only occurs in tumoral cell membrane,
(c) active serine proteases destroy cell surface of tumoral cells, they have apoptotic effect, which are the substantial findings on which this invention is based.
One of the characteristics of the invention is thus yet not described integration of lipase enzyme into anti-neoplastic composition and its wide-spectral efficiency newly distributed and confirmed by us.
Effect of Mixture of Protease Proenzymes and Hydrolytic Enzymes
The importance of in vivo application of protease proenzymes instead of active proteases is based on the existence of plasma anti-proteases, which together with proteases form complexes that prevent protease penetration to tumoral cell surface (Currie, G. A., Bagshawe, K. D., Lancet 279 (7492), 1967, 708-10). These anti-proteases are particularly alpha-1 antitrypsin and alpha-2 macroglobulin (Lah, T. T. et a., Expert Opin. Biol. Ther., 6, 2006, 257-279), for which proenzymes are illegible. The anti-tumoral selective effect of proenzymes of pancreatic proteases is based on presence of pancreatic secretory inhibitor (PSTI) formed by pancreatic acinar cells, which protect it against autodigestion. This inhibitor was also found on the surfaces of mucous membrane cells of stomach, small and large intestines, on adenoma cells, but not on carcinoma cells (Bohe, H. et al., J. Clin. Pathol., 43,1990, 901-904).
Nonparticipation in hemocoagulation cascade and invasive character of malignant tumours is another advantage of proenzyme application. Thanks to trypsin activity of malignant tumours protease proenzymes are selectively activated on tumour cells. This property of proenzymes circumventing the anti-protease protective effect does not affect tumour cells in vitro, where it is unimportant whether active proteases or their proenzymes are used. By in vitro trials we have demonstrated aggregative impact on tumour cells and by further in vivo trials also inhibitive impact on tumoral proliferation and metastases in several crucial lines of human tumour cells.
Finding that cells that did not respond succumbed to apoptosis was important (Trnka F. et al., EU Pat. 0743 070 A2, 1996; Novak, J. F., Trnka, F., Chernin, M. I., AACR Meeting Abstracts, Apr. 2006; 1023-1024.) Further authors described the suppressive role of trypsin to tumour progression by epigenetic mechanism (Yamashita, K. et al., Cancer Res., 63, 2003, 6575-6578), or anti-proliferative effects of strong alpha-amylase to mouse or human cancer cells by mediation of cellular adhesion and stimulation of cellular anoikis of apoptosis type (Fedrowitz, M. et al., J. Exp. Clin. Cancer Res., 30, 2011, 102-114). Elevation of plasmatic level of endostatin and angiostatin, and thus limitation of vascular neoplasm was also proven.
Amylase effect on tumour cells is thus being studied again as well as the role of trypsinogen and chymotrypsinogen (Itkonen, O., Scandin. J. Clin. Lab. Invest., 70, 2010, 136-143; Koskensalo, S. et al.: Oncology 82, 2012, 234-241) and effect of lipases (Nomura, D. K. et al., Cell 140, 2010, 49-61), namely triacylglycerol hydrolases, EC 3.1.1.3 as serine proteases, whose combined use is the principle of the effect of the four-composition according to this invention application.
The anti-metastatic effect of the therapeutic, diagnostic and prophylactic composition according to the invention is newly extended and boosted compared to the above state of the art by addition of vegetable lipase (Aub, J. C, Tieslau, C., Natl. Acad. Sci. USA, 50, 1963, 613-619), characterized at the active point by a triad of histidine-asparagine amino acids.
Therapeutic effect was demonstrated in nu/nu mice both in subcutaneous and rectal administration of the preparation in standardized trials with subcutaneously transplanted lines of human cells of mamma carcinoma, colorectal carcinoma, prostate cancer and small-cell lung cancer.
Today, we assign the universal anti-tumoral effect of the composition regardless of the origin of the affected tumours to the presence of sialized molecule of glycoprotein choriogonadotropin (Currie, G. A., Bagshawe, K. D., Brit. J. Cancer 22, 1968, 848-853; Willey, K. P. et al., J. Biol. Chem., 264, (1989), 1971, 619-729; Acevedo H. F. et al., Cancer 69, 1992, 1818-1928; Acevedo H. F. et al., Cancer 78, 1996, 2388-99), which we consider to be the target structure for chymotrypsinogen (Regelson W., Cancer 76, 1995, 1299-1301; Illes R. K.: Mol. Cell. Endocrinol., 260-262, 2007, 264-270) and amylase (Varki, N. M., Varki, A., Lab. Invest. 87, 2007, 851-857). This sialized centre is probably the carrier or mediator of the invasive and metastatic properties of malignant tumours (Nguyen, D. H., Tangvoranuntakul, P., Varki, A., J. Immunol., 2005; 175, 228-236).
Innovative application of lipase has been backed by recent finding of D. K. Nomura et al. who demonstrated experimentally that monoacylglycerol lipase (MAGL) regulates creation of free fatty acids in cancer cells, which enables them to create oncogenic lipidic signalling that increases migration, invasiveness of tumoral cells, tumour growth and pathogenicity. They found by means of proteomic approach based on analysis of tens of superordinate serine hydrolases that the MAGL levels are permanently increased right in the cells of aggressive tumours and they probably also pass aggressiveness to non-aggressive cells (Nomura, D. K., Long, J. Z., Niessen, S., et al., Cell 140, 2010, 49-61).
The wide-spectral anti-neoplastic effect of the new composition is given by its new structure, which accepts the complex relations in living organism of humans or animals. It does not prevent occurrence of tumour cells, however it destroys tumour cells that have already appeared, which restores and maintains complex balances of biological environment in normal healthy condition, limits occurrence and propagation of oncogenic signals.
Displays of the therapy based on anti-neoplastic composition administration like perspiration, breathing, urination and excrements of specific smell are also interesting and practically applicable. It is moreover accompanied by exhaustion, even somnolence or pains in muscles. All these signs are important, subjectively and clinically readable displays of the medicine contact with tissue affected by tumour. They actually do not indicate toxicity of the preparation as such but toxicity of products resulting from its impact on tumour cells. In this sense these signs also represent an important indicator whether tumour tissue is present in the organism or not. Unless any of the above signs occur after two or three days of administration of usual initial therapeutic doses of the composition according to the application there is very high degree of certainty that tumour cells are not present in the individual. In other words, with minimum toxicity, the composition according to the invention may be used in the above sense for diagnostics of tumour disease in asymptomatic period in individuals who have not noticed any disease symptoms yet.
In this relation we should mention the possibility of the use of haemodialysis or actually haemoperfusion, which may be applied if necessary in administration of such doses of the composition according to the invention that might threaten important life function of the patient as a subsequence of tumour cells destruction. Such an approach certainly requires specialist guidance and supervision of a clinical oncologist.
The principle of prophylactic utilization of the anti-neoplastic composition according to the invention may be basically described analogically. With regard to the minimum own toxicity of the preparation a period of administration of therapeutic doses of the composition may be included in the process of patient oncology monitoring. If tumour cells and thus the substrate for the activity of the preparation components were present in the organism the preparation administration would in fact already provide its effect prophylactically with the above accompanying symptoms.
The combined diagnostic and prophylactic effect of short-term administration of the composition thus also appears in an individual in whom no signs of oncology disease have occurred yet. Unless any of the above described symptoms occurs after preparation administration this fact can be logically considered an indicative proof of oncologic health.
The solution according to the invention is based on composition of two proenzymes of trypsinogen and chymotrypsinogen group with alpha-amylase and lipase (hereinafter also collectively referred to as active substances) defined from activity point of view. This four-composition shows in in vivo conditions surprisingly substantial positive effects towards wide spectre of tumour cells of completely different histological characteristics, as mentioned above, both after injection subcutaneous administration and non-invasive transmucosal, particularly rectal administration.
Partial Components of the Anti-Neoplastic Composition
The composition according to the invention represents a combination of enzymes and proenzymes obtained by extraction from organs (tissues) of animals (particularly mammals), plants and/or substances produced by cultivation methods using moulds and microorganisms or e.g. by continuous perfusion of mammal cells and consequent supernatant processing. With improvement of separation and analysis methods the proportion of cultivation process products is growing.
At today's level of knowledge in the field of biotechnology it is practically possible to obtain the appropriate proenzymes (Jungo, C., Marison, I., von Stockar, U.; J., Biotechnol., 128, 2007, 824-837; Paulová, L. et al., J. Biotechnol., 157, 2012, 180-188) and both the appropriate enzymes, i.e. all the necessary active substances of the composition by both the basic approaches.
Production procedures usually lead to production of isolated proteins and polypeptides, which are carefully taken from their natural environment, separated and identified. Contaminants originating from natural material may affect therapeutic, diagnostic and prophylactic use of proteins and polypeptides. They may contain not only protein components, but also numerous contaminants of various characters. They may lead to change of the original natural protein composition particularly to its glycosylation, secondary change or change of higher supramolecular structures, which might lead to undesirable immune or other reactions when administered to living organism. It is considered proven nowadays that not only the primary structure of proteins decides on their final interactions in organism, but obviously particularly the secondary and tertiary structures of proteins, which are in direct physical and chemical interactions with biological environment.
Because of the fact that any small changes of conditions may cause undesirable results mainly in cultivation procedures, very detailed and costly control of production processes, but also quality of final products is necessary. We know that this is also why the price of biologics is in average more than twenty times higher than the costs of medicines obtained by methods of conventional low-molecule chemistry or extraction and purification processes from organs and tissues of commonly accessible animals and plants.
It is thus necessary to define not only their original primary, secondary and tertiary structures, but for their further use also the acceptable deviations from the models of proteins and polypeptides in their original natural state. This is why not only requirements for purity are defined, e.g. 95% or preferably even 99% (depending also on chosen purity assessment method), acceptable residuum quantities, e.g. on N-terminal termination of the protein of protein amino acid internal sequence or amino acid chain glycosylation, but also acceptable percentage difference of the structure of those parts of protein or polypeptide that are important for the effect.
On the other hand we should stress that it has already been proven that changes in enzyme primary structure, e.g. in serine proteases do not have to lead to a change of enzyme function if the supramolecular structure of enzyme active part is preserved (Kraut, J., Annu. Rev. Biochem., 46, 1977, 331-58).
Next, but as well important fact is, that even primary amino acid sequence although it is authentic with the human protein model, does not guarantee immunocompatibility, as proven e.g. for erythropoietin (Prabhakar, S. S., Muhlfelder, T., Clin. Nephrology 47, 1997, 331-335) or interferon-α2B (Öberg, K. et al., J. Natl. Cancer Inst. 81, 1989, 531-535).
Important consequences for intended use of enzymes follow from the above facts. It is generally accepted that in most cases of enzyme distribution in system circulation and their behaviour in organism are controlled by combination of their size, charge, position and inclusion of hydrophilic surface functional groups. Information exists how these properties affect link to proteins, behaviour in circulation, interaction with vascular endothelial cells, extravasal capillary beds, on distribution by means of tissue stroma and final communication with target cells. However unifying principles describing what is the best for protein medicine particles, particularly in living organisms under in vivo conditions, do not exist yet. From this point of view the structures for targeted creation of pharmaceuticals are still too complex and the existing knowledge of their interaction mechanisms is still insufficient.
Even in the context of tumour diseases we should always remember that activity of enzymes as well as proenzymes expressed upon arbitrarily defined methods does not have to correspond to anti-tumour activity of the individual active substances.
It is definitely necessary to define biologic products, proenzymes and enzymes correctly and reproducibly. This is why definition of the composition according to the invention is not based on weight, but on enzymatic activity units.
Importance of this approach is obvious e.g. from comparison of two commercially available proenzymes from two different suppliers: chymotrypsinogen with declared activity>40 m.u./mg vs. chymotrypsinogen A with activity=1,422.3 m.u./mg (http://www.sigmaaldrich.com/catalog/product/sigma/t1143?lang=en®ion=CZ; http://www.applichem.com/en/home-page/). This is why the requirement for definition of biologic activity is so crucial for usability of information on biological pharmaceutical composition, e.g. in the form of activity per weight unit (usually milligram) per given volume or per one dose. This way of stating enzymatic activity is still used even by renowned manufacturers although in the world of science expression of enzymatic activity in catals (cat) as units derived from the SI system has been considered correct for a long time (1 U: 60=μcatal; 0.01667 U=μcatal and 60 μcatal=U).
An important difference between the composition according to the invention and earlier patents by Trnka, F. et al. or Psaledakis N. G. (Psaledakis N. G: U.S. Pat. No. 4,514,388, 1985), also inspired by J. Beard's findings is also based on this aspect. This difference is also essential in comparison with analogous patents or patent applications by Kenoyn, J. N. et al. based on the same idea which is therein hidden by heterogenic declaration of further claims related to various antioxidants and further potentially cancerostatic substances of heterogenic character. (Kenyon J. N. et al., Austral. Pat Appl. 2010310887, 2012; U.S. patent application Ser. No. 13/502,917, 2012; EP 2490711 A1, 2012). Moreover definition of the composition of the enzymatic preparations the last two mentioned patents refer to cannot in fact be based on weight of its substances, but on their enzymatic activity.
Production procedures for substances of the composition according to the invention including usual final lyophilization or spray drying use technology knowledge now well described and available in extensive scientific and patent literature. They not only enable obtaining high purity non-immunogenic products, but also products that may be modified unlike their natural models. Some of these modifications are aimed at improvement of some properties of their models, mostly stability properties or pharmacokinetic parameters. The other side of this progress and recently even a problem is the fact that routine production of modification of proteins, enzymes and polypeptides serves to circumvention of existing patent protection of the original substances, their compositions and preparations or actually abuse of the so called biosimilars way. The competent legislation authorities are trying to find and they gradually find consensus in this field in cooperation with scientific community. Data necessary for characterization of biological medicines, and also protein therapeuticals have been gradually formulated for biosimilars in directives EMA (Doc. Ref. EM EA/CHMP/BMWP/14327/2006.
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/W C500003946.pdf (on line 30.10.2013) and FDA Draft http://www.fda.gov/Drugs/Guidance ComplianceRegulatoryInformation/Guidances/default.htm. (on line 30.10.2013), the approach of the two regulatory authorities is somehow different, FDA is more reserved.
It is thus necessary to point out that each proenzyme (zymogene) and enzyme of our proposed anti-neoplastic composition may be obtained in tens of different was nowadays, some of which will naturally have the same effect as the substance used and tested by us and some will have not. Experts in the appropriate disciplines know that tens of substances belonging to the same enzyme classification group according to EC number have different primary structures, i.e. different numbers and orders of amino acids in chains, different numbers and positions of disulphide bridges, hydrophobic and hydrophilic parts of different lengths and locations, different places for substitution, different particular substituents, e.g. different sugar units glycosidically bound at different places and thus for example different behaviour in water environment, behaviour on interfaces, they may have different positions of active places for reaction with substrate. All that is given by the origin and by the method of production of the individual zymogens and enzymes.
Data and characteristics from scientific databases NCBI (http://www.ncbi.nlm.nih.gov/protein, on line 30.4.2013); and PDB http://www.rcsb.org/pdb/home/home.do, on line 30.4.2013) were used for specification of definition of proenzymes and enzymes of the anti-neoplastic composition according to this patent application.
The individual substances of the anti-neoplastic composition can be described by these characteristics and the extent of similarity of possible variations within that the same effects of both the partial components and the anti-neoplastic composition as a whole can be subsequently defined.
We thus consider important for the composition according to the invention that similarity of the particular variants of zymogens and enzymes therein used is roughly qualitatively specified by sequences of amino acids of the four tested substances (see Examples 2.1, 3.1., 4.1 and 5.1). In terms of the effect we should consider similar substances with at least 70% (and higher) correspondence of active places and sections of primary structures for the individual components of the four-composition. i.e. 70% correspondence for trypsinogen type I from bovine pancreas, for trypsinogen A from bovine pancreas, for α-amylase produced by Bacillus sp., and the same 70% correspondence for lipase from Tritici aestivum.
It is still true that no matter how important the enzyme amino acid sequence and its primary structure are, the enzyme participates in the effect itself in its supramolecular structure with particular positions of active places. The other parts of the enzyme are important in given context for its pharmacokinetics.
Necessary stabilization of the anti-neoplastic composition for the purpose of its elaboration into the pharmaceutical preparation is based on stabilization of the individual components. These components have to be assessed and evaluated as non-immunogenic for processing to the usable preparation and its administration. Their stabilization for processing and application is solved by using excipients and procedures that from the point of view of the present state of technology ensure preservation (or regeneration) of secondary and higher supramolecular structures of the partial active components and thus the four-composition according to our application as a whole (Lee, G.: Spray drying of proteins, in: Carpenter, J. Manning M. (Eds.), Rational Protein Formulation: Theory and Practice. Plenum Press, New York, 2002, 135-158). This is particularly application of procedures of processing solutions or dispersions of proteins by lyophilisation, spray lyophilisation, supercritical drying, possibly combination of dialysis and spray drying or cryogenic dispergation methods. If necessary, these methods are combined with application of a preparation from structurally stabilizing substances, particularly saccharides, e.g. sorbitol, trehalose, sucrose (Maury, M. et al., Eur. J. Pharm. Biopharm., 59, 2005, 251-261), polymers, e.g. polyethylene glycols, polyvinylpyrrolidones, dextrans (Platz, R. et al.: U.S. Pat. No. 6,019,468, 2000), lipids, e.g. medium-chain triglycerides (Hauss, D. J., Adv. Drug Deliv. Rev.; 59, 2007, 667-76; Tan, A., Rao, S., Prestidge, C. A.; Pharm. Res., 2013, 1-25), selected surfactants e.g. dipalmitoylphosphatidylcholine, polysorbates, polyoxyethylene stearates (Mansour, H. M., Damodaran, S., Zografi, G.: Mol. Pharm., 5, 2008, 681-695), cryoprotectants, e.g. glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide; hydroxyethyl starch, polyvinylpyrrolidone (Meryman H. T.; Cryobiology 8, 1971, 173-183).
Mixture of polyethylene glycols (PEGs) of adequate purity e.g. stabilizes the active substances physically-chemically and is also suitable for processing the active substances into application structures both for non-invasive administration types and parenteral administration. Stabilization effects of glycerol and n-propanol have been similarly described and are usable. The solution of the anti-neoplastic mixture according to the invention in given context thus uses information published on purification, regeneration a stabilization of supramolecular structures, namely for all partial components of the composition according to the invention (Pellegrini-Malpiedi, L., Picó, G. A, Nerli, B. B., Separ. Purif. Technol., 78, 2011, 91-96; Porfiri, M. C. et al.: Int. J. Biol. Macromol., 49, 2011, 7-13; Bassani, G. et al.; J. Chromatogr. B, 859, 2007, 222-228).
Administration Methods and Ways of Application
There is a known and generally acceptable fact that oral administration of polypeptide therapeutics and proteins to living organism of a human or animal body through the gastrointestinal tract represents a basic problem that has not been even partially solved yet. The conditions in stomach or intestines might actually completely destroy such medicine or prevent its absorption in the active form. This is why parenteral application is dominantly used nowadays for administration of biologics or therapeutic proteins, to which the composition according to the invention belongs.
Composition of the mixture and its dosage may be modified in details according to chosen application way and consequently also according to the vehicle used for the particular active substance, and dosage in clinical conditions may be defined and optimized according to the current state of the tumour disease or according to organism response to administered dose. The anti-neoplastic composition according to the invention basically enables outpatient treatment approach. It moreover enables self-administration particularly when non-invasive administration methods are used, and use of corresponding preparation types e.g. for sublingual or rectal administration. We consider both these ways advantageous for administration of the composition according to the invention as well as inhalation method through lung alveolar walls. Good tolerance to the preparation and absence of allergic symptoms or other negative immunological or other biological responses of the patient are certainly the basic conditions.
Parenteral administration with all its alternatives brought by the latest technological development in this field may obviously be applied to administration of the anti-neoplastic composition according to the invention. Dry injections, including lyophilized, i.e. mixtures of pulverized forms of active substances and auxiliary substances modifying pH, osmolarity, wettability, solubility, antioxidant protection, in the case of lyophilized products also excipients of cryoprotectant type (glycerol, dimethyl sulfoxide), lyoprotectants and structural substances. These preparations are dispersed in suitable liquid vehicle at the time of use and administered by injection needle.
Injection preparations with prolonged or otherwise modified active substance release, which release the active substance slowly and for longer period after administration, where it is desirable, e.g. in intraperitoneal application may also be used. Further excipients, of which both slowly degraded lipids and biodegradable polymeric systems based on proven polymers or oligomers of glycol acid, lactic acid and their co-polymers (Chaubal, M. V. et al.: Excipient selection and criteria for injectable dosage forms. In: Kathdare, A., Chaubal, M. V.: Excipient Development for Pharmaceutical, Biotechnology and Drug Delivery Systems. InformaHealthcare, New York, London 2006, 271-290; Gokarn, Y. R. et al, Excipient for protein drug. 291-331. In: Kathdare A., Chaubal M. V.: Excipient Development for Pharmaceutical, Biotechnology, and Drug Delivery Systems. InformaHealthcare, New York, London 2006, 291-331) are used for depot administration.
Administration though alveolar epithelium, which is the most penetrable and in terms of useful area of the alveoli absolutely the largest mucosal surface, should be the most suitable from this point of view. Inhaled low molecular medicine may appear in system circulation in seconds, which is very close to intravenous administration. This trait of inhalation application is however not important for the composition according to the invention. The effect start time in seconds or minutes is not important in this case, and for macromolecular substances it is unachievable. The second, but more important factor in this relation is the fact that penetration of inhaled particles through the complicated tree of branching bronchi and bronchioles up to the alveolar surface has already been satisfactorily solved. Newer findings show that unlike in bronchodilatation and antiasthmatic drugs the optimum size of solid particles for systemic administration of dry pulverized medicines of protein type is somehow lower, namely 2 to 4 micrometers (Patton, J. S., Byron, P. R., Nat. Rev. Drug Discovery 6, 2007, 67-74). This requirement may already be technologically met nowadays. The issue is thus passed to technical sphere of particle engineering and inhalator technical solution. The backward movement of particles from the alveoli (Scheuch, G. et al., J. Aerosol Med. Pulm. Drug Deliv., 23(S2), 2010, 39-5; Forbes, B. et al., Adv. Drug Deliv. Rev., 63, 2011, 69-87). However, physiologically desirable, remains to be solved from biopharmaceutical point of view. This is probably apart from commercial reasons one of the causes of insufficiently reproducible inhalation administration of insulin in the past, which has already been solved by inhalator innovation and immediately also by carrier free modification of insulin (Balduci, A. G. et al., Eur. J. Pharm. Sci., 2013, http://dx.doi.org/10.1016/j.ejps.2013.08.009, on line 18 Sep. 2013).
The composition according to the invention meets requirements for processability into the inhalation powder form both without further excipients and with suitable carrier (e.g. trehalose, lactose, mannitol), polyethylene glycol type substance of lower and medium molecule weight (e.g. macrogol 300 or macrogol 1500) and their mixtures. Inhalation of water dispersion of the composition according to the invention prepared at the time of application using nebulisers (e.g. electric or ultrasound) from ex tempore prepared water dispersions with the content of the above excipients, but also dextrans and suitable tensides (e.g. polysorbates) might have the same effect. The composition according to the invention is also suitable for processing into lipid micro particles, where their stabilizing and handling advantages and particularly advantageous properties as application systems for protein inhalation administration (Chow, A. H. L. et al., Pharm. Res. 24, 2007, 411-437; Mehnert, W., Mider, K.: Adv. Drug Deliv. Rev. 64, 2012, 83-101) may become useful.
As various practically applicable methods of application of inhalation (transalveolar) administration of the composition according to the invention exist from the point of view of possible technologies for production of particles suitable for inhalation from the point of view of physics and stability, we present one of them adopted from literature (Byrappa, K., Ohara, S. Adschiri, T., Adv. Drug Deliv. Rev., 60, 2008, 299-327), in Example 12 of embodiment of cryogenic procedure of creation of particles of anti-tumour composition for inhalation.
Analogous statement also applies to formulation-technological aspect of the individual partial methods of parenteral administration. We particularly applied subcutaneous injection administration of those documented in the application. Other partial methods of parenteral administration of the composition according to this patent application e.g. intraperitoneal, intrathecal are further possible considered variants (Huynh, G. H., Deen, D. F., Szoka, F. C., J. Control. Rel., 110, 2006, 236-259). They are well manageable from formulation-technological point of view, however the exact immunological properties of all the substances used, i.e. immunologic purity of the components of the composition according to the invention and of course the appropriate biologic response will be decisive for their use.
The reasons for the above stressed non-invasive methods of administration of the composition according to the invention are except for others based on the facts that both sublingual and rectal mucous membranes represent substantially permeable and also well accessible biological barriers, through which even multimolecular medicines penetrate. The speeds of medicine effect commencement are completely satisfactory for the intended anti tumour effect. It is moreover well known that substances in this administration methods do not suffer from quick first-pass effect in the absorption biological membrane itself (unlike in the intestine wall) after absorption.
We consider the fact that we have found a combination of the anti-neoplastic composition and application vehicle that in rectal administration in in vivo experiments on nu/nu mice anti-tumour effect against a chosen tumour line, namely line A 549 of small-cell lung tumour showed significantly better results than in administration of the same doses of the same composition by subcutaneous injection, a real innovative finding. Transport of substances to the tumour affected place is probably more complicated after subcutaneous administration.
The solution proposed by us linked at present with sublingual administration method, which in principle enables as fast (or slightly faster) commencement of medicine effect as subcutaneous injection administration, is relatively advanced. Its basic advantage is that it ensures slower and longer transfer to the system circulation to substances with large molecule (e.g. 50,000). We can thus actually talk about sublingual infusion. Such sublingual administration has a substantial advantage against parenteral, including subcutaneous administration. This is the fact that substances after absorption through sublingual mucous membrane are not taken by the bloodstream directly to the liver, where they are mostly metabolised and deactivated, but they avoid the liver first-pass effect. The path and time of their movement in organism are thus much longer and enable non metabolised substances to reach to more distant parts of the body. This is why we consider sublingual method very advantageous also for administration of the anti-neoplastic composition according to the invention.
Quickly disintegrating and soluble sublingual tablets or quickly soluble lyophilised tablets, the technology of which is well described and practically applied, can be generally used for sublingual administration. This issue with further application forms like sprays, gels, pastes, plasters, films and strips is dealt with in details by the latest summary by V. Hearnden (Hearnden V. et al., Adv. Drug Deliv. Rev., 64, 2012, 16-28). In the case of biologics the swallowing reflex and inhibition of medicine by saliva content are the main problems of application of this method.
A brand new type of sublingual preparation (new dosage form) suitable for biologics thanks to its composition and properties is based on application of nanofibre membranes (Stránská, D. et al.: Pat CZ 303 244; 2012). By their design and by using suitable pharmaceutically acceptable polymers they help avoid usual problems of other sublingual products, particularly their interaction with saliva or swallowing substantial part of the medicine. These nanofibre membranes have excellent mechanical properties, they usually enable up to 50 percent of active substances, including biologics, to be incorporated directly into the fibres. Even higher weight percentage of active substances may be anchored by impregnation. In mass production they are advantageously produced e.g. by means of electrospinning. They usually use carrying and structural polymers from the group of polyvinyl alcohol, polylactide, polycaprolactone, polyvinylpyrrolidone, their copolymers, copolymer polyethylene glycol/polyvinyl caprolactam/polyvinyl acetate, cellulose derivates like e.g. hydroxypropyl cellulose, hydroxypropyl methylcellulose, dextranes of various molecular weights, isolated as well as in mixtures and also numerous polymerized monomers of mixtures of at least two of them. A variant where the active substances are integrated directly in the nanofibre material of the preparation active layer designed for the contact with mucous membrane, which is covered by another nanofibre layer, e.g. polyurethane (see Example 12) protecting the active substances against contact with saliva is the most important for the composition according to the invention.
Moreover, and this may be substantial, deposition of biologics in nanofibres is probably today's most prospective way of biologics stabilization. Compared to the demandingness of cryogenic techniques and spray technologies this method is considerate from the point of view of the minimum temperature and pressure load on the active substance and from the point of view of integration of its structure in the fibre carrier. (Klein, S. et al., Biomacromolecules 10, 2009, 1751-1756). Thanks to mass production technologies of electrostatic spinning the anti-neoplastic composition according to the invention may be factually processed not only into nanomembranes for direct application of the active substances, but also into stabilized intermediate product and biologics type products for stock, all that actually without loss of biological activity of the active substances (e.g. of protein, polypeptide, virus or bacteria type).
The rectal administration method provides the same advantages in terms of first-pass effect avoidance and longer transport range for nonmetabolised therapeutic molecules. Except for minor problems, which are absolutely negligible with regard to the sense and importance of rectal administration of the anti-neoplastic combination according to the invention, this method can be definitely considered advantageous as we have repeatedly demonstrated for the composition according to the invention by the above mentioned in vivo trials.
Rectal systemic administration of medicines is traditionally used, well proven, backed by relatively wide selection of excipients and suitable manufacturing technologies. It may use conventional excipients, i.e. non-ionic substances from the group containing neutral lipids, e.g. tri-, di- or monoesters of higher fatty acids and polyols, e.g. glycerol, polyoxyethylene glycerides, e.g. polyoxyethylene glycol glyceryl-cocoate, polyoxyethylene glycols, polyethylene glycol ethers and higher fatty alcohols, e.g. lauryl alcohol, polyoxyethylene polar oils, e.g. ricin oil, oil saccharo-glycerides, polyethylene oxide and propylene oxide copolymers and their mixtures, if necessary with selected anti-oxidants (e.g. tocopherols, ascorbic acid, their derivatives like tocopheryl ascorbate) and further auxiliary substances.
“Co-administration” principle can be used to reach the effective levels of the anti-neoplastic composition in the organism. This means administration of all the composition parts in such amounts that ensure effect at the same time with regard to the application method or at different moments by means of one application preparation or more application preparations. This may be a single “co-administration” or multiple administrations at particular intervals. With the possibility to use various methods of administration a part of the composition may be administered by one of the possible methods (e.g. rectally) and the other part necessary to reach effective levels of the composition in the organism may be administered by different method, e.g. sublingually or parenterally.
Innovativeness
The essential innovation of the solution according to the invention is based on yet not described integration of lipase enzyme into an anti-neoplastic composition and its proven wide-spectral effectiveness. The newly designed composition of the preparation shows in in vivo trials a sum of activities bringing a complex, surprisingly strong cellular anti-tumoral effects. This finding is based on the results obtained from in vivo trials on nu/nu mice with subcutaneously implanted cells of standardized lines of human mamma carcinoma, colorectal carcinoma, pancreatic carcinoma and small-cell lung carcinoma.
Further innovation of the invention is in the composition definition based on enzymatic activity units, not on weight proportions of the individual active substances as it is in earlier remotely similar patents (Trnka et al, 1996, 1998, see above). This approach to efficiency definition ensures technical feasibility of the invention and reproducibility of composition of the active substances, and thus also of the appropriate manners designed for their administration to organism. Today's pharmacopoeias include requirement for using the activity characteristics for enzymes.
Further innovation of the solution according to the invention is in the fact that we have demonstrated and applied upon the achieved results advantageous non-invasive methods of administration and appropriate dosage systems for administration of the enzyme and proenzyme composition. Unlike in all the existing patents dealing with administration of biologics we have proposed preparations for non-invasive transmucosal administration in wide scale of indications of the anti-tumoral composition according to the invention. Surprisingly positive therapeutic results of rectal administration showed us the way to sublingual administration. This brings prospect of improved compliance for a patient and possibility of self-administration of the anti-neoplastic preparation.
Innovation of the solution is also in the fact that we define suitable vehicles for administration of the composition in direct relation to the chosen application way and also definite therapeutic, diagnostic or prophylactic requirements. Particularly polyethylene glycol of appropriate molecule weight or a mixture of selected polyethylene glycols (e.g. macrogol 300 and macrogol 1500 in weight ratio 45:55) or glycerol, N-propanol or trehalosa and further saccharides (sucrose, mannitol) as structural stabilizers for proteins are advantageous excipients for the active substances individually as well as for their compositions. The solution according to the invention also considers application of polyethoxylated lipidic substances (e.g. stearoyl polyoxyl-6-glycerid), neutral lipids (e.g. glyceryl-palmitostearate), esters of monovalent alcohols with higher fatty acid (e.g. isopropyl-myristate, isopropyl-palmitate) in processing anhydrous pulverized anti-tumoral composition and permeation enhancers, e.g., glycerol, cyclopentadecanolide, polycarbophil-cysteine.
Innovation of the solution is also in preparation and use of the above structural and stabilizing excipients as well as the technology of electrostatic spinning for processing the individual components into a stabilized composition of proenzymes and enzymes as a formulating intermediate product (see Example 11, Example 12).
1. Amylasa: Alpha-amylasa from Bacillus sp. Type II-A, lyophilised powder. Isolated from Bacillus amyloliquefaciens. Sigma-Aldrich. Prague. Product No.: A 6380; EC No. (Sigma): 232-560-9; EC No.: 3.2.1.1; CAS No.: 9000-90-2
Molecular weight: 58,403
Activity: 1,333 m.u./mg of solid substance; 3,100 m.j/mg of protein 2. Lipase: Lipase from wheat germ, Type I; lyophilised powder. Isolated from Triticium aestivum. Sigma-Aldrich. Prague. Product No.: L 6380, EC No. (Sigma): 232-619-9
EC 3.1.1.3; CAS No.: 9001-62-1
Molecular weight: 143,000
Activity: 5-15 m.u./mg of protein
3. Chymotrypsinogen: α-Chymotrypsinogen A from bovine pancreas. lyophilised powder, without salt content. Applichem. Prague. Product No.: A069
CAS No.: 9035-75-0
Molecular weight: approx. 25,000
Activity: min. 1,200 m.u./mg
4. Trypsinogen: Trypsinogen from bovine pancreas. Dialyzed and lyophilised powder, without salt content. Sigma-Aldrich. Prague. Product No.: T1143; EC No. (Sigma): 232-651-3; CAS No.: 9002-08-8
Molecular weight: 23,700
Activity: 10,900 m.u./mg of protein
The individual components of the composition can be processed according to usual rules as powder mixture and then as an intermediate product towards the required dosage form. Individual finely ground components may also be gradually integrated to a prepared vehicle or its part for the purpose of primary processing with suitable carrier (e.g. trehalosa for injection administration, see Example 12, stabilizing excipient (e.g. n-propanol, polyethylene glycol 300) or complete vehicle (hardened fat with added isopropyl-myristate as suppository base) according to particular intended application (see Example 8 and Example 9) In processing and in possible storage of the compositions requirements of the manufacturers of the individual components have to be kept. (e.g. temperature, humidity, protective atmosphere, ambient purity).
Specific quantity of the therapeutic composition required for administration of one dose to a human depends on characteristics of the particular individual (weight, age, health condition parameters including individual reactivity to the administered composition), anti-neoplastic disease characteristics (e.g. type, location, stage) administration method (e.g. systemic sublingual, parenteral infusion, systemic rectal), way of application (e.g. monotherapeutic, sequential, graded) and preparation physical character (e.g. colloid solution, separated powder mixture).
Examples 2.1, 3.1, 4.1, and 5.1 represent sequences of amino acids of enzymatic and proenzymatic substances for anti-neoplastic compositions according to Example 1 and Table 1. Related reference examples 2.2, 2.3, 3.2 a{hacek over (z)} 3.4, 4.2, and 5.2 represent sequences of amino acids of representatives of biologically similar substances that may be used as substitutes for substances according to Table 1 as so called “biosimilars” namely individually or in the complete structure. Maintaining the effect quality the individual enzymatic activities of the components in relation to protein weight unit are always important for such biologically similar compositions prepared this way. This characteristic is one of the factors deciding on suitability of an alternative “biosimilar” composition for a particular or considered application.
The examples in principle illustrate the possibility of substitution of partial substances, both, by biologically similar isolated natural substances or biotechnologically produced substances.
Example 2.1 Sequence of Amino Acids of Alpha-Amylase, Bacillus Species, Amyloliquefacienshttp://www.brenda-enzymes.org/index. php4?page=sequences/seq.ph p4?ID=7605 (on line 18 Sep. 2013) (further see Table 1)
http://www.uniprot.org/uniprot/M8AC56 (on line 18 Sep. 2013)
http://www.uniprot.org/uniprot/P06278 (on line 18 Sep. 2013)
http://www.uniprot.org/uniprot/Q8L5T0 (on line 18 Sep. 2013)
http://www.uniprot.org/uniprot/P00591 (on line 18 Sep. 2013)
http://www.uniprot.org/uniprot/Q6F357 (on line 18 Sep. 2013)
http://www.uniprot.org/uniprot/C6A8G0 (on line 18 Sep. 2013)
Chain A:
http://www.ncbi.nlm.nih.gov/protein/1TGN_A (on line 18 Sep. 2013)
Chain A
http://www.ncbi.nlm.nih.gov/protein/2CGA_A (on line 18 Sep. 2013)
Chain B
http://www.ncbi.nlm.nih.gov/protein/2CGA_B (on line 18 Sep. 2013)
http://www.brenda-enzymes.org/index.php4?page=sequences/seq.php4?ID=7605 (on line 18 Sep. 2013)
Formula (g) for 100 doses
The mixture is prepared as mixture lyophilised powder containing Composition 1 as per Table 1 with structural stabilizing trehalose, subsequently aseptically distributed in 100 vials. Preparation packing contains an ampoule with water vehicle of this content (mg/100 ml):
Water vehicle is for ex tempore preparation of 3 millilitres of solution from dry lyophilised powder. Appropriate dose of Composition 1 in the resulting solution of volume 3 millilitres is then applied by droplet infusion of suitable composition e.g. with dextran 10,000.
Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.
Example 7 Preparation with Anti-Neoplastic Composition for Injection Subcutaneous Administration for Human Mamma Carcinoma TreatmentFormula (g) for 100 doses
The mixture is prepared as mixture lyophilised powder containing Composition 1 as per Table 1 and stabilizing polyethylene glycol 4000 and subsequently aseptically distributed in 100 vials. Preparation packing contains an ampoule with solution of 8 mg of sodium chloride in 1 ml of water for injection.
Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.
Example 8 Preparation with Anti-Neoplastic Composition for Human Colorectal Carcinoma Treatment for Rectal Administration by Lipophilic SuppositoryFormula (g) for 100 suppositories
-
- 1. Stirring slowly in a suitable vessel melt mixture of hydrogenated glycerides from coco oil to about 35° C. producing homogenous dispersed phase.
- 2. Mix gradually the same weight quantity of tempered isopropyl-myristate or stearoyl-polyoxyl-6-glyceride to well homogenized mixture of Composition 2 according to Table 1 in another vessel once producing a concentrated premix.
- 3. Stirring slowly add gradually the melted lipophilic dispersed phase to the homogenized premix.
- 4. Continue stirring at temperature below 35° C. for at least 15 minutes, then still slowly stir and let it cool to temperature between 30° C. and 31° C.
- 5. Then pour the suppository mass with the content of anti-neoplastic composition to prepared suppository mould forming suppositories of about 1.8 grams each.
- 6. Continue stirring the suppository substance when pouring into the forms to prevent the composition from sedimentation but not aerating the melt. Protective atmosphere may be used if necessary.
The above Composition 2 is administered as a hydrophobic suppository in one morning dose.
Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.
Example 9 Preparation with Anti-Neoplastic Composition for Human Small-Cell Lung Carcinoma Treatment for Rectal Administration by Hydrophilic SuppositoryFormula (g) for 100 suppositories
-
- 1. Stirring slowly in a suitable vessel melt mixture of polyethylene glycol 300 and polyethylene glycol 1500 to 40° C. producing homogenous dispersed phase.
- 2. Mix gradually the same weight quantity of n-propanol or glycerol to well homogenized mixture of Composition 2 according to Table 1 in another vessel producing concentrated suspension.
- 3. Stirring slowly add gradually the melted hydrophilic dispersed phase to the homogenized suspension.
- 4. Continue stirring at temperature below 35° C. for at least 15 minutes, then let it cool to temperature between 30° C. and 31° C. under slow stirring.
- 5. Then pour the suppository substance with the anti-neoplastic Composition 2 to prepared suppository mould of forming suppositories about 1.8 grams each.
- 6. Continue stirring the suppository substance when pouring into the forms to prevent the composition from sedimentation but not aerating the melt.
The above Composition 2 according to Table 1 is administered as a hydrophilic suppository in one morning dose, or in a half dose in the morning and half dose at noon.
Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.
Example 10 Preparation with Anti-Neoplastic Composition 3 for Human Pancreatic Carcinoma Treatment for Sublingual AdministrationFormula of nanofibrous membrane (g) for 100 applications
-
- 1. Prepare concentrated mixture of Composition 3 according to Table 1 with glycerol buffered to pH 7.4 in a suitable vessel.
- 2. Prepare solution of trehalose, polyethylene oxide 400 and hydroxypropyl methyl cellulose in water vehicle in another vessel.
- 3. Add gradually the trehalose solution as per 2 to the spread of Composition 3, stir thoroughly and put it in the supply bin of the manufacturing device NS WS 50 (Elmarco, Liberec, CZ).
- 4. Ionic composition of the bin content is optimized for electrospinning process.
- 5. After the check of conductivity and process parameters spin the prepared solution at temperature not exceeding 45° C. producing nanofibrous membrane that is anchored on the base material belt.
- 6. In the next cycle relay the nanofibrous membrane in analogical process from the solution of hydroxymethyl propyl cellulose and polyethylene glycol 400.
- 7. Form the combined two-layer nanomembrane according to the produced square weight of Composition 3 in nanofibrous membrane and according to the required dose, namely to strips of 10 cm2 each.
- 8. The strip determined for single administration is adjusted in a storing part of suitable packaging.
The nanofibrous sublingual preparation is administered in the morning and in the evening after meal as adhesive film on the bottom side of tongue.
Specific dosage of the composition is based on complex oncologic examination of particular individual.
Example 11 Preparation with Anti-Neoplastic Composition 3 for Human Pancreatic Carcinoma Treatment for Sublingual AdministrationFormula of nanofibrous membrane (g) for 100 applications
Procedure: Analogous to Example 9. Relaying of the nanofibrous reservoir is performed by electrospinning of water insoluble polyurethane.
The resulting two-layer preparation is applied by the protective polyurethane layer towards the mouth cavity and by the hydroxypropyl methyl cellulose reservoir of Composition 3 to the sublingual side.
The nanofibrous sublingual preparation is administered in the morning and in the evening after meal as adhesive film on the bottom side of tongue.
Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.
Example 12 Preparation with Anti-Neoplastic Composition 4 for Human Small-Cell Lung Carcinoma Treatment for Inhalation AdministrationFormula (g) of powder for 100 inhalations
-
- 1. Solve the weight quantity of Composition 4 in 100 g 20% (weight) water solution of trehalose.
- 2. Put a magnetic mixer in isolated dispergation vessel and cover with conic cover with closable portholes.
- 3. Insert an ultrasonic probe (120 kHz) in the vessel through one of the portholes and attach the vessel to the magnetic mixer table.
- 4. After filling the vessel up to the edge with liquid nitrogen put the cover on the vessel and let the liquid still.
- 5. Squirt approximately 5 ml of the solution on the nitrogen surface and close the cover.
- 6. Drive 3 ml/min. of water solution of the anti-neoplastic composition 4 and trehalose to the work space by a peristaltic pump through another porthole and switch on the magnetic mixer.
- 7. After dispergation transfer the produced solid particles of the solution to Class I clear glass vials and close them provisionally with lyophilizing plug at normal temperature.
- 8. Put the vials on partitions of the freeze-dryier (GFT 6, Klein Vakuumtechnik), Niederfishbach, DE) and reduce pressure to 80 kPa.
- 9. Cool the vials gradually to 0° C. for 3 hours, then to −35° C. for 12 hours, primary drying is performed by temperature increase to −10° C. for 8 hours and to 10° C. for 8 hours.
- 10. After temperature rise to 30° C. during 1 hour secondary drying continues at 30° C. for 6 hours at pressure 10 kPa.
- 11. After tempering the product to normal temperature fill the freeze-dryier with sterilized air and close the vials with the lyophilised product.
The obtained powder is prepared for processing, filling and application in dose powder inhaler (e.g. of Turbhaler, Easyhaler, Novolizer, Certihaler type) or as pressurized powder (e.g. in Ultrahaler or MAG-haler type inhalers), or in a single-dose system with pre-adjusted powder capsules (e.g. Spinhaler, Aerolizerk, Handihaler), or powders in multi-dose capsule or blister systems (e.g. Diskhaler or Diskus).
Example 13 Preparation with Anti-Neoplastic Composition 4 for Human Laryngeal Carcinoma Treatment for Inhalation Administration by NebuliserPowder formula (g) for nebulisation of 10 doses (g):
The powder composition for reconstitution for inhalation is aseptically distributed in ten glass injection bottles to 100 ml while the content of Composition 4 is 48.2 mg.
For the reconstitution 1 bottle is filled with water for injection or sterilized water. Nebulisation is performed in a suitable small e.g. jet based, vibrating membrane or electronic nebuliser of e.g. Spag-2, PARI LC Star, Aero-Eclipse or Pro-Dose type.
Example 13 Preparation for Intraperitoneal Administration for Mamma Carcinoma Treatment in a Dog or a CatFormula (g) for 100 doses
The above Formula is processed as mixture lyophilised powder containing Composition 5 according to Table 1 and structural stabilizing trehalose distributed into 100 vials.
It is administered in one dose as eutonic-isotonic water solution after reconstitution ex tempore from dry powder in 5% glucose solution.
Specific dosage of the composition is based on complex oncologic examination of particular individual.
Claims
1. Anti-proliferative and anti-metastatic pharmaceutical composition containing a mixture of proenzymes and enzymes, wherein the composition consists of following active substances: proenzyanes trypsinogen and chyrmotrypsinogen, and enzymes α-amylase and lipase, wherein the ratio of enzymatic active substances, namely activities of trypsinogen (T), chymotrypsinogen A (CH), α-amylase B.s (A) and lipase T.a. (L) for T:CHI:A:L ratio expressed in m.u. is in the range from 150:150:40:1 to 400:1200:200:1, and further comprises one or more pharmaceutically acceptable excipients, for simultaneous, separate and subsequent administration of the composition in parenteral or transmucosal way, while the composition is for therapeutic, prophylactic and anti-metastatic use in mammals.
2. Pharmaceutical composition according to claim 1, wherein the trypsinogen is of type I, chymotrypsinogen is of type A, α-amylase is produced by Bacillus sp. and lipase is from Triticum aestivum.
3. Pharmaceutical composition according to claim 1, wherein the minimum enzymatic activity of active substances is as follows: trypsinogen 40 m.u./mg, chymotrypsinogen 60 m.u./mg, α-amylase 20 m.u./mg and lipase I m.u./mg.
4. Pharmaceutical composition according to claim 1, wherein at least one of the active substances is replaced with biologically similar active substance obtained by extraction from higher plants, animals or by cultivation procedures using mould cells, yeast cells, or bacteria, the primary structure of the biologically similar substance with the active substance which it has replaced in the composition being at least 70% identical and the position of active places essential for the effect is at least 95% identical.
5. Pharmaceutical composition according to claim 1, wherein the composition is for systemic sublingual, rectal, inhalation or parenteral administration.
6. Pharmaceutical composition according to claim 1, wherein it contains as the pharmaceutically acceptable excipients: one or more hydrophilic polyhydric alcohols;
- hydrophilic low molecular alcohols;
- saccharides;
- polysorbates; poloxamers;
- one or more lipophilic excipients;
- esters of higher fatty acids with glycerol or propylene glycol;
- esters of lower monovalent alcohols;
- esters of higher fatty acids with medium and higher fatty alcohols;
- higher fatty alcohols and analogously higher fatty acids
- vegetable oils;
- phospholipids;
- sterols;
- biocompatible and biodegradable polymers
- or any combination thereof.
7. Pharmaceutical composition according to claim 1 designed for sublingual administration, wherein it is in the form of nanofibres, while it contains
- at least one of polyvinyl polymers like polyvinylpyrrolidone with molecular weight approx. 30,000 to 50,000 and polyvinyl alcohols with molecular weight from 20,000 to 200,000, of cellulose derivatives like methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose and/or polysaccharides of starch type like hydroxyethyl starch, carboxymethyl starch sodium salt and/or dextrins with molecular weight from 4,000 to 80,000,
- and/or of biotechnological polysaccharides of dextran type with molecular weight from 10,000 to 80,000,
- and/or glucuronate type substances like xanthan mucilage, and/or further polyuronides or their salts, particularly sodium, potassium, like hyaluronans, alginans, pectinans, arabinans and/or polymers based on acrylic, methacrylic acids and/or their copolymers like carboxyvinyl polymers (carbomers) cross-linked with s polyalkenyl ethers of sugars or poly alcohols (like diallyl sucrose a diallyl penta erythritol, biodegradable polyesters of α-hydroxy acids like (PDLLA), (PGA), (PLGA), polycaprolactones with molecule weight from 10,000 to 100,000, further polymeric excipients of copolymer type like polyvinyl caprolactam-polyvinyl acetate polyethylene glycol.
8. Pharmaceutical composition or its part according to claim 1 designed for inhalation administration, wherein it contains at least one or more saccharides, including trehalose, mannitol, glucose and/or various forms of lactose.
9. Pharmaceutical composition according to claim 1, wherein it is in the form of nanofibre stabilized preparation for direct administration of active substances or as stabilized storage of active substances in an intermediate product or in the final preparation.
10. The pharmaceutical composition according to claim 6, wherein the hydrophilic polyhydric alcohol includes polyethylene glycol with a molecular weight of between 100 to 8,000.
11. The pharmaceutical composition according to claim 6, wherein the hydrophilic low molecular alcohol is selected from glycerol, propylene glycol, n-propanol, or any combination thereof.
12. The pharmaceutical composition according to claim 6, wherein the saccharide is selected from trehalose, mannitol, lactose, sorbitol, myoinositol, or any combination thereof.
13. The pharmaceutical composition according to claim 6, wherein the polysorbate is selected from polysorbate 20, polysorbate 60, polysorbate 80, or any combination thereof.
14. The pharmaceutical composition according to claim 6, wherein the poloxamer is selected from poloxamer 182, poloxamer 417, poloxamer 908, or any combination thereof.
15. The pharmaceutical composition according to claim 6, wherein the lipophilic excipient includes hydrogenated triglycerides selected from hydrogenated glycerol trioleate, hydrogenated glycerol cocoate, or any combination thereof.
16. The pharmaceutical composition according to claim 6, wherein the esters of higher fatty acids with glycerol or propylene glycol are selected from glycerol tripalmitate, glycerol trioleate, glycerol tristearate, glycerol distearate, glycerol dioleate, glycerol monolaurate, propylene glycol myristate, glycerol dipalmitostearate, or any combination thereof.
17. The pharmaceutical composition according to claim 6, wherein the esters of lower monovalent alcohol is selected from diisopropyl adipate, isopropyl laurate, isopropyl linoleate, isopropyl palmitate, or any combinations thereof.
18. The pharmaceutical composition according to claim 6, wherein the esters of higher fatty acids with medium and higher fatty alcohols include myristyl stearate, capryl stearate, cetyl palmitate, caprin behenate, lauroyl oleate, or any combination thereof.
19. The pharmaceutical composition according to claim 6, wherein the higher fatty alcohol is selected from lauryl alcohol, myristyl alcohol, palmityl alcohol, stearyl alcohol, behenyl alcohol and the analogous higher fatty acids is selected from lauric, myristic, palmitic, stearic, lignoceric, arachidonic, behenic acids and their ethoxylated derivatives, selected from polyethylene glycol 10 oleyl alcohol, polyethylene glycol 25 stearyl alcohol, polyethylene glycol 40 stearyl alcohol, stearoyl polyethylene glycol 32 glycerol, polyethylene glycol 15 hydroxy stearate, or any combination thereof.
20. The pharmaceutical composition according to claim 6, wherein the vegetable oil is selected from cottonseed oil, sunflower oil, groundnut oil, soya oil, castor oil, and their ethoxylated derivatives selected from polyoxyl 35 ricinoleate, or any combination thereof.
21. The pharmaceutical composition according to claim 6, wherein the phospholipids are selected from egg lecithin, soya lecithin, dioleoylphosphatidylcholine, dipalmitoylphosposphatidylserine, or any combination thereof.
22. The pharmaceutical composition according to claim 6, wherein the sterols are selected from cholesterol and its derivatives selected from cholesteryl linoleate, cholesteryl acetate, or any combination thereof.
23. The pharmaceutical composition according to claim 6, wherein the biocompatible and biodegradable polymers are selected from polyesters selected from poly-DL-lactic acid (PDLLA), polyglycolic acid (PGA), poly-DL-lactic glycolic acid (PLGA), or any combination thereof.
Type: Application
Filed: Nov 24, 2014
Publication Date: Nov 24, 2016
Inventors: Frantisek TRNKA (Ceske Budejovice), Pavel DOLEZAL (Hradec Kralove)
Application Number: 15/037,331
