PHARMACEUTICAL COMPOSITION CONTAINING A MIXTURE OF PROENZYMES AND ENZYMES

Abstract

Pharmaceutical composition containing a mixture of proenzymes and enzymes, containing proenzymes trypsinogen and chymotrypsinogen and enzymes -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.

Claims

1. A method for treating or preventing cancerous tumors, metastases, or both, wherein said method comprises administering a pharmaceutical composition containing a mixture of proenzymes and enzymes, wherein the composition consists of following active substances: proenzymes trypsinogen and chymotrypsinogen, and enzymes -amylase and lipase, wherein the ratio of enzymatic active substances, namely activities of trypsinogen (T), chymotrypsinogen A (CH), -amylase produced by Bacillus species (-amylase B.s, A) and lipase produced by Triticum aestivum (lipase T.a., L) for T:CH:A:L ratio expressed in i.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.

2. The method according to claim 1, wherein the trypsinogen is of type I.

3. The method according to claim 1, wherein the minimum enzymatic activity of active substances is as follows: trypsinogen 40 i.u./mg, chymotrypsinogen 60 i.u./mg, -amylase 20 i.u./mg and lipase 1 i.u./mg.

4. The method 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, wherein the amino acid sequence of the biologically similar active substance is at least 90% identical to the amino acid sequence of the active substance that was replaced with the biologically similar active substance.

5. The method according to claim 1, wherein the composition is for systemic sublingual, rectal, inhalation or parenteral administration.

6. The method 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. The method 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, a lginans, 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 a-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. The method 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. The method 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 method according to claim 6, wherein the hydrophilic polyhydric alcohol includes polyethylene glycol with a molecular weight of between 100 to 8,000.

11. The method according to claim 6, wherein the hydrophilic low molecular alcohol is selected from glycerol, propylene glycol, n-propanol, or any combination thereof.

12. The method according to claim 6, wherein the saccharide is selected from trehalose, mannitol, lactose, sorbitol, myoinositol, or any combination thereof.

13. The method according to claim 6, wherein the polysorbate is selected from polysorbate 20, polysorbate 60, polysorbate 80, or any combination thereof.

14. The method according to claim 6, wherein the poloxamer is selected from poloxamer 182, poloxamer 417, poloxamer 908, or any combination thereof.

15. The method 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 method according to claim 6, wherein the esters of higher fatty acids with glycerol or propylene glycol are selected from glycerol tripa Imitate, glycerol trioleate, glycerol tristearate, glycerol distearate, glycerol dioleate, glycerol monolaurate, propylene glycol myristate, glycerol dipalmitostearate, or any combination thereof.

17. The method 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 method 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 method 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 oeyl 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 method 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 method according to claim 6, wherein the phospholipids are selected from egg lecithin, soya lecithin, dioleoylphosphatidylcholine, dipalmitoylphosphatidylserine, or any combination thereof.

22. The method 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 method 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.

Description

DESCRIPTION OF FIGURES

[0094] FIG. 1 shows a part of photo documentation visually comparing the treated and untreated mouse with subcutaneously transplanted MDA-MB-231 line of mamma carcinoma in 36-day in vivo trial in everyday rectal administration of the anti-neoplastic composition. Particularly, FIG. 1 presents a photo documentation of in vivo trial on nu/nu mice with MDA-MB-231 line of mamma carcinoma in 36-day rectal administration of composition K2 (dose 2, lipophilic vehicle) compared to the reference mouse (without administration).

[0095] FIG. 2 shows graphic interpretation of average values of tumour volumes (including SD) in 36-day in vivo trial on nu/nu mice with subcutaneously transplanted MDA-MB-231 line of mamma carcinoma in everyday subcutaneous and rectal administration of the anti-neoplastic composition according to this application (composition K1, or K2 according to Table 1); female nu/nu mice; approx. 28 g; 8 mice in each group; lipophilic suppository vehicle; dose 2 represents double quantity of K2 composition. Particularly, FIG. 2 presents the average values of tumour volumes (including SD) during 40-day in vivo trial with MDA-MB-231 line in subcutaneous and rectal administration of the anti-neoplastic composition 2 (female nu/nu mice; approx. 28 g; 8 mice in each group; dose 2 contains double quantity of composition 2).

[0096] FIG. 3 shows a part of photo documentation visually comparing the treated and untreated mouse with subcutaneously transplanted H 116 line of colorectal carcinoma in 75-day in vivo trial in everyday rectal and subcutaneous administration of the anti-neoplastic composition. Particularly, FIG. 3 presents a photo documentation of the first third of in vivo trial on nu/nu mice with H 116 line of colorectal carcinoma in everyday rectal administration of composition 2 (dose 1; lipophilic base) compared to the reference mouse (without administration).

[0097] FIG. 4 shows graphic interpretation of average values of tumour volumes (including SD) in 75-day in vivo trial on nu/nu mice with subcutaneously transplanted H 116 line of colorectal carcinoma in everyday subcutaneous and rectal administration of the anti-neoplastic composition according to this application; female nu/nu mice; approx. 28 g; 8 mice in each group; lipophilic suppository vehicle. Particularly, FIG. 4 presents the average values of tumour volumes (including SD) up to the 23.sup.rd day of in vivo trial on nu/nu mice with H 116 line of colorectal carcinoma in subcutaneous and rectal administration of the anti-neoplastic composition K1 (K2) (female nu/nu mice; approx. 28 g; 8 mice in each group).

[0098] FIG. 5 shows a part of photo documentation visually comparing the treated and untreated mouse with subcutaneously transplanted CAPAN 2 line of pancreatic carcinoma in 85-day in vivo trial in everyday subcutaneous and rectal administration of the anti-neoplastic composition. Particularly, FIG. 5 presents a photo documentation of in vivo trial with CAPAN 2 line of pancreatic carcinoma on nu/nu mice in 85-day of subcutaneous rectal administration of the composition K2 compared to the reference mouse (without administration).

[0099] FIG. 6 shows graphic interpretation of average values of tumour volumes (including SD) in 85-day in vivo trial on nu/nu mice with subcutaneously transplanted CAPAN 2 line of pancreatic carcinoma in everyday subcutaneous and rectal administration of the anti-neoplastic composition according to this application; female nu/nu mice; approx. 28 g; 8 mice in each group; lipophilic suppository vehicle. Particularly, FIG. 6 presents the average values of tumour volumes (including SD) v in vivo trial on nu/nu mice with CAPAN 2 line of pancreatic carcinoma in everyday subcutaneous and rectal administration of anti-neoplastic composition K1, or K 2; (female nu/nu mice; approx. 28 g; 8 mice in each group).

[0100] FIG. 7 shows a part of photo documentation visually comparing treated and untreated mouse with subcutaneously transplanted A 549 line of small-cell lung carcinoma on the 40th and 85th day of in vivo trial in everyday administration of the anti-neoplastic composition. Particularly, FIG. 7 presents a photo documentation of 75-day (99-day) in vivo trial with A 549 line of small-cell lung carcinoma on nu/nu mice in everyday rectal administration of composition 2 (hydrophilic base, dose 2) compared to the reference mouse (without administration).

[0101] FIG. 8 shows graphic interpretation of average values of tumour volumes (including SD) in an in vivo trial with subcutaneously transplanted A 549 line of small cell lung carcinoma in everyday rectal administration of the anti-neoplastic composition (K2 according to Table 1) female nu/nu mice; approx. 28 g; 8 mice in each group; hydrophilic suppository vehicle. Particularly, FIG. 8 presents the average values of tumour volumes (including SD) in 75-day (99-day) in vivo trial on nu/nu mice with A 549 line of small-cell lung carcinoma in everyday subcutaneous and rectal administration of anti-neoplastic composition K1, resp. K2 (female nu/nu mice; approx. 28 g; 8 mice in each group; dose 2 is double quantity of dose 1).

EXAMPLES OF EMBODIMENT

Example 1

Qualitative Structure of the Anti-Neoplastic Composition According to the Invention Application

[0102] 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

[0103] Molecular weight: 58,403

[0104] Activity: 1,333 i.u./mg of solid substance; 3,100 m.j/mg of protein

[0105] 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

[0106] EC 3.1.1.3; CAS No.: 9001-62-1

[0107] Molecular weight: 143,000

[0108] Activity: 5-15 i.u./mg of protein

[0109] 3. Chymotrypsinogen: -Chymotrypsinogen A from bovine pancreas. lyophilised powder, without salt content. Applichem. Prague. Product No.: A069

[0110] CAS No.: 9035-75-0

[0111] Molecular weight: approx. 25,000

[0112] Activity: min. 1,200 i.u./mg

[0113] 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;

[0114] CAS No.: 9002-08-8

[0115] Molecular weight: 23,700

[0116] Activity: 10,900 i.u./mg of protein

TABLE-US-00001 TABLE 1 Examples of proportional weight combination of parts of the anti-neoplastic composition for formulation of the preparations for various administration methods. Qualitative composition is in Example 1. Identification Composition 1 Composition 2 Composition 3 Composition 4 Composition 5 Method of Administration Subcutaneous Rectal Sublingual Inhalation Intraperitoneal Quantity mg mg mg mg mg Amylase 2.07 4.07 5.6 1.95 9.6 Lipase 2.89 9.89 12.8 9.89 12.2 Chymotrypsinogen 10.24 14.24 14.24 28.5 14.2 Trypsinogen 29.58 22.32 29.58 14.24 35.0

[0117] 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)

[0118] 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).

[0119] 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).

[0120] 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 az 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.

[0121] 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

[0122] Sequence of amino acids of alpha-amylase, Bacillus species, Amyloliquefaciens (further see Table 1)

TABLE-US-00002 1 MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAVNGTLMQYFEWYTPNDGQHWKRLQNDAEH 61 LSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLH 121 SRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQETSEEYQIKAWTDFRFPGRGNTYS 181 DFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYADVDYDHPDVV 241 AETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVAEYWQNNAGK 301 LENYLNKTSFNQSVEDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDT 361 QPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILK 421 ARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETW 481 YDITGNRSDTVKIGSDGWGEFHVNDGSVSIYVQ

Reference Example 2.2

[0123] Sequence of amino acids of biologically similar (90%) -amylase, Triticum urartu (Red wild einkorn), (Crithodium urartu)

TABLE-US-00003 1 MERRGLLKAALLASCLLVVCSGRVPTVIQQPSTTIYNSTLAKTLVEYAAAIYTADLTQLF 61 TWTCDRCGDLIEGFEMMDIIVDVESCLEAYVGFASDINAVVVVFRGTQENSIQNWIEDLL 101 WKQLDLDYPGMPEAMVHRGFYSAYHNTTIRDGIVSGIQKTQKLHGDVPIMVTGHSMGAAM 151 ASFCALDLVVNYGLDDVKLMTFGQPRVGNAAFASYLKRYLPHAIRVTNANDIVPHLPPYF 201 SFFPQKTYHHFPREVWVHDVGLGSLVYTVEQICDDSGEDPACSRSVSGNSIQDHITYLGV 301 SMHAEAWSSCRIVMDYAELRYKMDLHGNVVLSKQQQQSGLSNERRRHSAQ

Reference Example 2.3

[0124] Sequence of amino acids of biologically similar (90%) -amylase, Bacillus lichenformis

TABLE-US-00004 1 MKQQKRLYARLLTLLFALIFLLPHSAAAAANLNGTLMQYFEWYMPNDGQHWKRLQNDSAY 61 LAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLH 121 SRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVISGEHRIKAWTHFHFPGRGSTYS 181 DFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNENGNYDYLMYADIDYDHPDVAAE 241 IKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWQNDLGALE 301 NYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNSTVVSKHPLKAVTFVDNHDTQP 361 GQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKAR 421 KQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHD 480 ITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR

Example 3.1

[0125] Sequence of amino acids of lipase, Tritici aestivum (see Table 1)

TABLE-US-00005 1 MERRGLLKTALLACLLVVCSGRVPMVIQQPSTTIYNSTLAKTLVEYAAAIYTADLTQLFT 61 WTCDRCGDLIEGFEMMDIIVDVENCLEAYVGFASDINAVIVVFRGTQENSIQNWIEDLLW 121 KQLDLDYPGMPEAMVHRGFYSAYHNTTIRDGIVSGIQKTRKLHGDVPIMVTGHSMGAAMA 181 SFCALDLVVNYGLDDVKLMTFGQPRVGNAAFASYFKRYLPHAIRVTNANDIVPHLPPYFS 241 FFPQKAYHHFPREVWVHDVGLGSLVYTVEQICDDSGEDPACSRSVSGNSIQDHITYLGVS 301 MHAEAWSSCRIVMDYAELRYKMDLHGNVVLSKQQQQQPGLSDQRRRHSAQ

Reference Example 3.2

[0126] Sequence of amino acids of biologically similar lipase, Sus Scrofa

TABLE-US-00006 1 SEVCFPRLGCFSDDAPWAGIVQRPLKILPWSPKDVDTRFLLYTNQNQNNYQELVADPSTI 61 TNSNFRMDRKTRFIIHGFIDKGEEDWLSNICKNLFKVESVNCICVDWKGGSRTGYTQASQ 121 NIRIVGAEVAYFVEVLKSSLGYSPSNVHVIGHSLGSHAAGEAGRRTNGTIERITGLDPAE 181 PCFQGTPELVRLDPSDAKFVDVIHTDAAPIIPNLGFGMSQTVGHLDFFPNGGKQMPGCQK 241 NILSQIVDIDGIWEGTRDFVACNHLRSYKYYADSILNPDGFAGFPCDSYNVFTANKCFPC 301 PSEGCPQMGHYADRFPGKTNGVSQVFYLNTGDASNFARWRYKVSVTLSGKKVTGHILVSL 361 FGNEGNSRQYEIYKGTLQPDNTHSDEFDSDVEVGDLQKVKFIWYNNNVINPTLPRVGASK 421 ITVERNDGKVYDFCSQETVREEVLLTLNPC

Reference Example 3.3

[0127] Sequence of amino acids of biologically similar lipase (50%), Oryza sativa Japonica Group

TABLE-US-00007 1 MSSSPMLGGIADRWRELHGQDSWNGLLDPLDLDLRSSILSYGELVQATYDSFNRERRSPH 61 AGACVYGHGDLLAAAGASAAGSYAVTKFVYATSGLPVPEAFLLLPLPSLLPPAWSRESNW 121 MGYVAVATDEGVAALGRRDIVVAWRGTVESLEWVNDFDFTPVPAAPVLGAAAAANPRAIV 181 HRGFLSVYTSSNKDSKYNKASARDQVLEEVRRLMELYKDEVTSITVVGHSLGASLATLNA 241 VDIVANGANCPPASSSSSQPPCPVTAIVFASPRVGDGFFKAAFASFPDLRALHVKNAGDV 301 VPMYPPLGYVDVAVKLRISTSRSPYLRSPGTIETLHNLECYLHGVAGEQGSAGGFKLEVD 361 RDVALANKGVDALKDKYPVPPRWWVSKNRCMVKDADGHWALHDFEQI

Reference Example 3.4

[0128] Sequence of amino acids of biologically similar lipase, Bifidobacterium animalis subsp. lactis DSM 10140

TABLE-US-00008 1 MELYRNNEIPPIEYTPGTSEFRDAVIGLARYWTAIAEDLHADEPGVQERTAAACLRFRKE 61 CAMFDYARALQWHDPQGVYVHTDIPYLPDGGYRDGEVRGHLLDVYIPRDAIVRGGNTLPV 121 YIDIHGGGFTYGYKELNRNFNTHLADLGFGVFSLNYRPAPQTDLVGQLHDIQAALCWIGE 181 HITQFPVSPDNIFITGDSAGACLSLLTLLIEHNDDAAHAFGIERASGIHLRGASLISGVY 241 DITPSSPMRARLAETVGNEFFAGLDDATVFLDPADWLTQGIGIPPLFLVTSSDDFVQSET 301 LALATSLARNGRDFELHDFKVPCTQTLGHVFPVGMTWLPESERVLHGIREFSYPLTR

Example 4.1

[0129] Sequence of amino acids of trypsinogen, Bos Taurus (see Table 1)

[0130] Chain A:

TABLE-US-00009 1 VDDDDKIVGGYTCGANTVPYQVSLNSGYHFCGGSLINSQWVVSAAHCYKSGIQVRLGEDN 61 INVVEGNEQFISASKSIVHPSYNSNTLNNDIMLIKLKSAASLNSRVASISLPTSCASAGT 121 QCLISGWGNTKSSGTSYPDVLKCLKAPILSDSSCKSAYPGQITSNMFCAGYLEGGKDSCQ 181 GDSGGPVVCSGKLQGIVSWGSGCAQKNKPGVYTKVCNYVSWIKQTIASN

Reference Example 4.2

[0131] Sequence of amino acids of biologically similar trypsinogen I Sequence 2, U.S. Pat. No. 7,049,484, 2006

TABLE-US-00010 1 CGVPAIQPVLSGLSRIVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAHCGV 61 TTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAASFSQTVSA 121 VCLPSASDDFAAGTTCVTTGWGLTRYTNANTPDRLQQASLPLLSNTNCKKYWGTKIKDAM 181 ICAGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCSTSTPGVYARVTALVNWVQQ 241 TLAAN

Examples 5.1

[0132] Sequence of amino acids of chymotrypsinogen A, Bos taurus (see Table 1)

[0133] Chain A

TABLE-US-00011 1 CGVPAIQPVLSGLSRIVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAHCGV 61 TTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAASFSQTVSA 121 VCLPSASDDFAAGTTCVTTGWGLTRYTNANTPDRLQQASLPLLSNTNCKKYWGTKIKDAM 181 ICAGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCSTSTPGVYARVTALVNWVQQ 241 TLAAN

[0134] Chain B

TABLE-US-00012 1 CGVPAIQPVLSGLSRIVNGEEAVPGSWPWQVSLQDKTGFHFCGGSLINENWVVTAAHCGV 61 TTSDVVVAGEFDQGSSSEKIQKLKIAKVFKNSKYNSLTINNDITLLKLSTAASFSQTVSA 121 VCLPSASDDFAAGTTCVTTGWGLTRYTNANTPDRLQQASLPLLSNTNCKKYWGTKIKDAM 181 ICAGASGVSSCMGDSGGPLVCKKNGAWTLVGIVSWGSSTCSTSTPGVYARVTALVNWVQQ 241 TLAAN

Reference Example 5.2

[0135] Sequence of amino acids of biologically similar chymotrypsinogen B (synthetic construct, CDS clone)

TABLE-US-00013 1 MAFLWLLSCWALLGTTFGCGVPAIHPVLSGLSRIVNGEDAVPGSWPWQVSLQDKTGFHFC 61 GGSLISEDWVVTAAHCGVRTSDVVVAGEFDQGSDEENIQVLKIAKVFKNPKFSILTVNND 121 ITLLKLATPARFSQTVSAVCLPSADDDFPAGTLCATTGWGKTKYNANKTPDKLQQAALPL 181 LSNAECKKSWGRRITDVMICAGASGVSSCMGDSGGPLVCQKDGAWTLVGIVSWGSDTCST 241 SSPGVYARVTKLIPWVQKILAAN

Reference Example 5.3

[0136] Sequence of amino acids of biologically similar chymotrypsinogen B, Gadus morhua

TABLE-US-00014 1 MGHEVDSVLPGLFRRTYGCGRPAISPVITGYSRIVNGEEAVPHSWSWQVSLQDQTGFHFC 61 GGSLINENWVVTAAHCNVKNYHRVVLGEHDRSSNSEGVQVMTVGQVFKHPRYNGFTINND 121 ILLVKLATPATLNMRVSPVCLAETDDVFEGGMKCVTSGWGLTRYNAADTPALLQQAALPL 181 LTNEQCKKFWGNKISDLMICAGAAGASSCMGDSGGPLVCQKAGSWTLVGIVSWGSGTCTP 241 TMPGVYARVTELRAWVDQTIAAN

Example 6

[0137] Preparation with anti-neoplastic Composition 1 for injection intravenous administration for human mamma carcinoma treatment.

[0138] Formula (g) for 100 doses

TABLE-US-00015 Composition 1 2.239 Trehalose 25.00

[0139] 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):

TABLE-US-00016 Hydrogen sodium phosphate, dihydrate 167 mg Potassium dihydrogen phosphate 20 mg Potassium chloride 20 mg Sodium chloride 800 mg Polysorbate 80 10 mg Polyethylene glycol 300 3.0 ml Water for injection to 100.0 ml

[0140] 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.

[0141] Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.

Example 7

[0142] Preparation with anti-neoplastic composition for injection subcutaneous administration for human mamma carcinoma treatment.

[0143] Formula (g) for 100 doses

TABLE-US-00017 Composition 1 2.580 (see Tab. 1) Polyethylene glycol 4000 5.160

[0144] 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.

[0145] Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.

Example 8

[0146] Preparation with anti-neoplastic composition for human colorectal carcinoma treatment for rectal administration by lipophilic suppository.

[0147] Formula (g) for 100 suppositories

TABLE-US-00018 Composition 2 2.563 Isopropyl-palmitate or 1.9 Stearoyl polyoxyl-6-glyceride Hardened fat 180.0

[0148] Procedure: [0149] 1. Stirring slowly in a suitable vessel melt mixture of hydrogenated glycerides from coco oil to about 35 C. producing homogenous dispersed phase. [0150] 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. [0151] 3. Stirring slowly add gradually the melted lipophilic dispersed phase to the homogenized premix. [0152] 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. [0153] 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. [0154] 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.

[0155] The above Composition 2 is administered as a hydrophobic suppository in one morning dose.

[0156] Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.

Example 9

[0157] Preparation with anti-neoplastic composition for human small-cell lung carcinoma treatment for rectal administration by hydrophilic suppository.

[0158] Formula (g) for 100 suppositories

TABLE-US-00019 Composition 2 2.563 n-propanol or glycerol 1.9 Polyethylene glycol 300 95.0 Polyethylene glycol 1500 85.0 [0159] 1. Stirring slowly in a suitable vessel melt mixture of polyethylene glycol 300 and polyethylene glycol 1500 to 40 C. producing homogenous dispersed phase. [0160] 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. [0161] 3. Stirring slowly add gradually the melted hydrophilic dispersed phase to the homogenized suspension. [0162] 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. [0163] 5. Then pour the suppository substance with the anti-neoplastic Composition 2 to prepared suppository mould of forming suppositories about 1.8 grams each. [0164] 6. Continue stirring the suppository substance when pouring into the forms to prevent the composition from sedimentation but not aerating the melt.

[0165] 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

[0166] Preparation with anti-neoplastic Composition 3 for human pancreatic carcinoma treatment for sublingual administration.

[0167] Formula of nanofibrous membrane (g) for 100 applications

TABLE-US-00020 Composition 3 3.11 Trehalosa 10.0 Glycerol 85% buffered to pH 7.4 3.5 Hydroxypropyl methyl cellulose 2.2 Polyethylene glycol 400 1.1 Redistilled water q.s.

[0168] Procedure: [0169] 1. Prepare concentrated mixture of Composition 3 according to Table 1 with glycerol buffered to pH 7.4 in a suitable vessel. [0170] 2. Prepare solution of trehalose, polyethylene oxide 400 and hydroxypropyl methyl cellulose in water vehicle in another vessel. [0171] 3. Add gradually the treha lose 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). [0172] 4. Ionic composition of the bin content is optimized for electrospinning process. [0173] 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. [0174] 6. In the next cycle relay the nanofibrous membrane in analogical process from the solution of hydroxymethyl propyl cellulose and polyethylene glycol 400. [0175] 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 cm.sup.2 each. [0176] 8. The strip determined for single administration is adjusted in a storing part of suitable packaging.

[0177] The nanofibrous sublingual preparation is administered in the morning and in the evening after meal as adhesive film on the bottom side of tongue.

[0178] Specific dosage of the composition is based on complex oncologic examination of particular individual.

Example 11

[0179] Preparation with anti-neoplastic Composition 3 for human pancreatic carcinoma treatment for sublingual administration.

[0180] Formula of nanofibrous membrane (g) for 100 applications

TABLE-US-00021 Composition 3 3.11 Mannitol 10.0 n-propanol buffered to pH 7.4 3.5 Polyvinyl alcohol 2.2 Polyethylene oxide 400 1.1 Polyurethane 0.9 Redistilled water q.s.

[0181] Procedure: Analogous to Example 9. Relaying of the nanofibrous reservoir is performed by electrospinning of water insoluble polyurethane.

[0182] The resulting two-layer preparation is applied by the protective polyurethane layer towards the mouth cavity and by the hydroxypropyl methyl celulose reservoir of Composition 3 to the sublingual side.

[0183] The nanofibrous sublingual preparation is administered in the morning and in the evening after meal as adhesive film on the bottom side of tongue.

[0184] Specific therapeutic, diagnostic or prophylactic dosage of the composition is based on complex oncologic examination of particular individual.

Example 12

[0185] Preparation with anti-neoplastic Composition 4 for human small-cell lung carcinoma treatment for inhalation administration.

[0186] Formula (g) of powder for 100 inhalations

TABLE-US-00022 Composition 4 2.73 Trehalose 20.00 Water for injection to 100.0

[0187] Procedure: [0188] 1. Solve the weight quantity of Composition 4 in 100 g 20% (weight) water solution of treha lose. [0189] 2. Put a magnetic mixer in isolated dispergation vessel and cover with conic cover with closable portholes. [0190] 3. Insert an ultrasonic probe (120 kHz) in the vessel through one of the portholes and attach the vessel to the magnetic mixer table. [0191] 4. After filling the vessel up to the edge with liquid nitrogen put the cover on the vessel and let the liquid still. [0192] 5. Squirt approximately 5 ml of the solution on the nitrogen surface and close the cover. [0193] 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. [0194] 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. [0195] 8. Put the vials on partitions of the freeze-dryier (GFT 6, Klein Vakuumtechnik), Niederfishbach, DE) and reduce pressure to 80 kPa. [0196] 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. [0197] 10. After temperature rise to 30 C. during 1 hour secondary drying continues at 30 C. for 6 hours at pressure 10 kPa. [0198] 11. After tempering the product to normal temperature fill the freeze-dryier with sterilized air and close the vials with the lyophilised product.

[0199] The obtained powder is prepared for processing, filling and application in dose powder inhaler (e.g. of Turbha ler, 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

[0200] Preparation with anti-neoplastic composition 4 for human laryngeal carcinoma treatment for inhalation administration by nebuliser.

[0201] Powder formula (g) for nebulisation of 10 doses (g):

TABLE-US-00023 Composition 4 0.482 g Trehalosa 5.50

[0202] 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.

[0203] 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

[0204] Preparation for intraperitoneal administration for mamma carcinoma treatment in a dog or a cat.

[0205] Formula (g) for 100 doses

TABLE-US-00024 Composition 5 2.239 (see Tab. 1) Trehalose 20.00

[0206] 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.

[0207] Specific dosage of the composition is based on complex oncologic examination of particular individual.