Patent Application
20190001000
Fluorescent conjugate of hyaluronic acid or salt thereof, hydrophobized conjugate, methods of preparation and use thereof.
The present invention relates to a fluorescent ester conjugate of hyaluronic acid or a salt thereof containing in vivo diagnosable heptamethine indocyanine dye (Cypate) of the formula III or 1-[3-(2-carboxyethyl)-1,1-dimethyl-5,9b-dihydrobenzo[e]indol-3-ium-2-yl(chloride)]-octa-1,3,5,7-tetraenyl]-1,1-dimethyl-2H-benzo[e]indol-3-yl]propanoic acid. Further it is described a hydrophobized conjugate, methods of preparation thereof and an use for in vivo imaging applications and a treatment of neoplasms.
Hyaluronic acid or a salt thereof (HA) is a linear polysaccharide belonging to the group of important glucosamino glycanes. In the terms of the structure, it is a biopolymer formed with repeated disaccharide units (β-(1→4) glycosidic bond), consisting of D-glucuronic acid and N-acetyl-D-glucosamine mutually linked with a β-(1→3) glycosidic bond (see formula 1 below).
Hyaluronic acid occurs in the physiological environment in the form of sodium salt as a very hydrophilic and highly hydrated biopolymer (Schanté C. E; et al., Carbohydr. Polym. 2011, 85 (3), 469-489). HA plays an important role in the structure and organization of the extracellular matrix and also forms suitable environment for cells, their proliferation, differentiation and mobility (D'Este M.; et al., Carbohydr. Polym. 2014, 108, 239-246; Schanté, C. E.; et al., F. Carbohydr. Polym. 2011, 85 (3), 469-489). HA is further contained in vertebrates in all body organs and the extracellular matrix of soft connective tissues (Eenschooten, C.; et al., Carbohydr. Polym. 2010, 79 (3), 597-605).
This, in the water soluble polysaccharide is relatively easily subjected to the chemical modification and it appears as a biomolecule very suitable for a conjugation with other compounds, including fluorescent agents. An advantage of HA is also the existence of several cellular receptors thereof, which can be used for specific aiming of derivatives, for example, into tumor tissues (Garg H. G.; et al., Chemistry and biology of hyaluronan, 1st ed.; Elsevier: Netherlands, 2004).
Recently, one of the suitable methods for noninvasive diagnostics, beside nuclear imaging technics, the roentgen radiography and the computer tomography, is the optical imaging through fluorescence. Fluorescence appears as a promising method for in vivo diagnostics, especially thanks to its relatively high sensitivity, specificity and image gaining in the real time (Ye, Y.; et al., Bioconjugate Chem. 2008, 19 (1), 225-234). Among advantages of the fluorescent imaging are also relatively low cost, feasibility, non-invasiveness and safety in comparison to the ionizing radiation. This technic is very perspective for the detection, diagnostics and prevention in the tumor therapy, but also as a complement method for gaining complex information in clinic applications in the imaging using the positron emission tomography (PET), SPECT (“single-photonemission computed tomography”) or MRI. (“magnetic resonance imaging”), (Ye, Y.; et al., Theranostics 2011, 1, 102-106).
In the application of the non-invasive diagnostic methods is the penetration of irradiation through tissues highly dependent on absorption properties and the refractive index (Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7 (5), 626-634). The absorption and emission wavelength in the area 650-900 nm, that is radiation in the near infrared area (NIR), are considered as optimal. In comparison with shorter wavelengths of visible spectra, the radiation of these wavelengths penetrates deeper and at the same time no absorption by the endogenous fluorophores and rise of undesired autofluorescence occurs (Kobayashi, H.; et al., Chem. Rev. 2010, 110, 2620-2640, Luo, S. et al.; Biomaterials 2011, 32 (29), 7127-7138).
Recently, cyanine fluorescent dyes, derivatives of squarine, phtalocyanines, porphyrines and also some agents derived from boron-dipyrromethen (BODIPY) (Luo, S.; et al., Biomaterials 2011, 32 (29), 7127-7138) belong among important exogenous contras agents.
Among very frequent agents used for imaging optical methods belong cyanine dyes. Structurally, they relate most to two heterocyclic structures, where one of these heterocycles carries positively charged nitrogen atom and they are further linked through the polymethine bridge, shown in the general formula of cyanine fluorescent agent, where
X—(CH═CH)n—CH═Y
X and Y are heterocyclic nitrogen structures and n=1-3. If n=1, dyes count among tricyanine, n=2 pentacyanine and n=3 heptacyanine compounds.
Generally, the length of the polymethine bridge determines fluorescent properties of the given derivative and with every accruing n occurs the rise of the absorption and emission wavelengths of the compounds by about 100 nm. The typical wavelengths for the trimethine dyes are about 500 nm, for pentamethine about 600 nm and heptamethine derivatives eventually rich wavelengths of the near infrared area. Bathochromic and hypsochromic shift of wavelengths is further partially influenced by the type of the heterocyclic structure that also determines absorption and radiance of the fluorescent dye (Hermanson, G. T. Bioconjugate Techniques, 2. ed.; Elsevier: United States of America, 2008).
Conjugates of NIR Fluorescent Agents with Various Molecules
In the past, it was possible to use NIR fluorescent agents alone only to nonspecific imaging, e.g. blood circuit and its purification (Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7 (5), 626-634). Polymethine cyanine dyes tend to an aggregate in the aqueous environment, which results in the loss of their fluorescence and it is therefore disadvantageous in in vivo diagnostics (U.S. Pat. No. 6,641,798 B2). The said fluorescent agents are further characterized by very the short halftime of the degradation in the circuit system (150-180 s), that limits accumulation of the dye in the examined target, for example, in the tumor tissue and by that decreases also in vivo contrast (Hill T. K. et. al., Bioconjug Chem. 2015; 26(2): 294-303). Simple and general approach how to increase the accumulation of the contrast agents in target places and tissues, or ensure the better solubility in the aqueous environment, is their conjugation with the suitable ligand from the group of peptides, proteins, saccharides etc. (Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7 (5), 626-634). The conjugation of the contrast agents to the carrier polymer or the nanoparticle is mostly performed through a linker. The linker should be used to ensure fluorescent (to prevent quenching) and/or degradable conditions of system (Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7 (5), 626-634). The disadvantage of this solution is that the reaction has more steps and it is very difficult, and derivatization of the entering agents is often used, which is disadvantageous in the term of the purification of the products and/or the isolation of the reaction intermediates is necessary, which is again disadvantageous. In the case of nanoparticle systems, the linker length has to be chosen so, that no fluorescence quenching of the contrast agent occurs and the contrast agent is in vivo detectable (i.e. the linker must not be too short) (US20110104070).
Patent document U.S. Pat. No. 6,641,798 posts the demands to the general structure of the low-molecular conjugates of the bioactive molecules (for example peptides, proteins, antibodies, saccharides) with the cyanine fluorescent agents, prepared to increase the detection and the therapy of the tumor. A serious disadvantage of used derivatives in in vivo application is the short stability of fluorescence (c. 45 min) at the imaging.
In literature there were also described conjugates of NIR fluorescent agent Cypate with the several different ligands (amino saccharides, peptides, polysaccharides) in term of the contrast agents for in vivo diagnostics. The preparation of NIR fluorescent agent Cypate itself is described in document WO2002032285, the improved synthesis was further published in the publication Ye, Y.; et al., Bioconjugate Chem. 2005, 16, 51-61. For in vivo studies prepared Yunpeng Ye.; et al. (Ye, Y.; et al., J. Am. Chem. Soc. 2004, 126, 7740-7741), polyvalent probes, where one or more monosaccharide units of D-(+)-glucosamine were bound on the carboxyl group of through the amide bond. Given NIR fluorescent agent formed the core of the dendrimeric formation and at the same time it served as a chromophore for nanoparticles, the biodistribution thereof was investigated using non-invasive optical methods in vivo and then ex vivo. One disadvantage of this solution, where no polymer was used for the conjugation, is insolubility of the system in the aqueous solutions in the absence of organic solvent.
Another type of known nanoparticle for tumor imaging is a dually targeted polymer micelle on the basis of succinyl chitosan with a covalently bond Cypate using the amide bond, further with methionine and folic acid (Chen, H.; et al., Polym. Chem. 2014, 5, 4734-4746). A disadvantage of this solution is using of chitosan as a carrier polysaccharide, as chitosan is a body-foreign agent. Another disadvantage is very limited solubility of the native chitosan in the physiological conditions, the nature chitosan must be therefore always modified, because otherwise it would not be possible to use chitosan in the intravenous application. Furthermore, the modified chitosan is usually limitedly soluble in the physiological conditions. The modification can cause changes in the biodegradability and biocompatibility of chitosan depending on the desired application (Balan, V.; et al., European Polymer Journal 2014, 53, 171-188; Dumitriu, S. Polymeric Biomaterials, 2nd ed.; Marcel Dekker, Inc.: United States of America, 2002). Chitosan conjugated with Pluronic F68 and with the cyanine dye (Cy5.5) was used in tracking the accumulation in the tumor mouse model (W. II Choi.; et al., Nanomedicine: Nanotechnology, Biology and Medicine 2015, 11, 359-368). Without targeting of the carrier system with herceptin was the tumor tracking imaging in vivo possible, however, after 12 hours the NIR fluorescence intensity did not continue to increase and it can be therefore supposed that this system does not enable the long term tracking of the tumor disease in vivo without the necessity of further carrier addition. The similar disadvantage of the short imaging time (maximum of 1-2 days after the application) is known also in the other polymer and non-polymer conjugates with NIR dyes (D. Kokuryo; et al., Journal of Controlled Release 2013, 178, 125, Tan X.; et al., Biomaterials 2012, 33, 2230-2239).
In literature, Cypate was further described as a part of multifunctional imaging probe, when on one carboxyl group of the said fluorescent agent was bound a chelating component of cation metals through the amide bond with the purpose of forming the optical-nuclear imaging system. Cypate was further conjugated to the cyclic RGD peptide with the purpose of targeting specific cells (Ye, Y.; et al., Bioconjugate Chem. 2008, 19, 225-234). The disadvantage of this system is that its fluorescent conditions change in the presence of metal cations and in some cases (presence of Fe3+ or Cu2+) can lead up to the complete quenching of fluorescence (Ye, Y.; et al., Bioconjugate Chem. 2008, 19, 225-234).
Conjugates of NIR Fluorescent Agents with Hyaluronic Acid
In publications Choi K. (Choi, K. Y.; et al., J. Mater. Chem. 2009, 19 (24), 4102-4107 and Choi, K. Y.; et al., Biomaterials 2010, 31 (1), 106-114), there was described a hydrophobized derivative of hyaluronic acid with 5β-cholic acid. However it was found that that the action of 5β-cholic acid leads to supporting of carcinogenesis of the intestine tumors in their early stages. (Baijal, K. P.; et al., Canadian Journal of Physiology and Pharmacology, 1998, 76(12), 1095-1102). Further, NIR fluorescent agent Cy5.5 was conjugated on the carboxyl group of hyaluronic acid, using ethyl-3(3-dimethylaminopropyl)-carbodiimide/hydroxy-benzotriazol. (EDC/HOBt) and linker dihydrazide of adipic acid. A disadvantage is not only multistep synthesis, but also used activator EDC/HOBt, that can cause undesirable intramolecular netting of hyaluronan and with that corresponding decrease of solubility of the final product (Huerta-Angeles, G.; et al., Carbohydr. Polym. 2014, 111 (13), 883-891). The derivative of hyaluronic acid conjugated with Cy5.5 through the amide bond was further used for the surface treatment of superparamagnetic nanoparticles iron oxides (SPION) with the purpose of forming multimodal probes for combination of in vivo MRI/optical detection of activity of hyaluronidase (Lee, D.; et al., Macromol. Res. 2011, 19 (8), 861-867). With respect to in vivo diagnostics through the optical method, the fluorescent agent Cy5.5 from the group of pentamethine derivatives is not the ideal choice. In contrast to heptamethine derivatives, they do not reach such high absorption and emission wavelengths, that are for in vivo non-invasive diagnostics recommended, that is in using wavelengths typical for pentamethine derivatives can create the undesirable autofluorescence of endogenous fluorophores.
In the case of heptamethine derivatives of cyanine is known the conjugate of hyaluronic acid with commercially available NIR fluorescent IR-783, modified four-step synthesis resulting in IR-783-S-Ph-COOH, where this derivative was consequently bound through the amide bond to hyaluronic acid. The derivative was prepared to study deeper understanding of pharmacokinetic of HA in vivo, its degradation and role in the physiological and pathological conditions (Wang, W.; Cameron, A. G.; Shi, K. Molecules 2012, 17, 1520-1534). Unfortunately, the very complicated synthesis is not transformable into the industrial scale either in terms of economic or in terms of very low yield (Wang, W.; Cameron, A. G.; Shi, K. Molecules 2012, 17, 1520-1534).
Among other representatives of this type of fluorescent agents belongs indocyanine green (ICG). HA conjugated with indocyanine green and polyethylene glycol (PEG) makes aggregated particles in the aqueous environment, with the possibility of in vivo dual imaging of the tumor using optical methods and the photoacoustic detection (Miki, K.; et al., Biomacromolecules 2015, 16, 219-227). The large disadvantage of using indocyanine green in vivo is hepatobiliary toxicity and fast removal of this dye by liver (G. R. Cherrick, et al. J. Clinical Investigation, 1960, 39, 592-600). The disadvantage of derivatives of HA with NIR fluorescent agents, when it comes to their conjugation onto the carboxyl group of hyaluronic acid and that in modification of the carboxyl function of HA, results in the neutralization of the natural negative charge of biopolymer, further can influence the solubility of HA in the physiological environment and not lastly also disturbs the recognition of HA by cell receptors (Mero, A.; et al., Carbohydr. Polym. 2010, 79 (3), 597-605).
The said disadvantages of the prior art are overcome by a fluorescent conjugate of hyaluronic acid or a salt thereof of the general formula I
wherein R+ is H+ or a physiologically acceptable salt selected from the group containing Na+, K+, Mg2+ or Ca2+,
R1 is —H or the cypate residue of the formula II, where is a place of covalent bond of a cypate residue of the formula II
one R1 being the cypate residue of the formula II in at least one repeated unit providing that if there is R1 the cypate residue of the formula II in the unit, then the other R1 in the unit are H,
and where n is an integer in the range of 2 to 625.
According to a preferred embodiment of the invention the cypate residue of the formula II is substituted at the position 6 of glucosamine part of the fluorescent conjugate of hyaluronic acid or the salt thereof of the general formula I.
Cypate I is bound to a hydroxyl group of hyaluronic acid (see Scheme 1 and 2, below) after the activation, which is advantageous especially in terms of maintaining the biological properties of hyaluronic acid and also of its solubility in the physiological environment. The solubility of the conjugate of the present invention is 1 to 3 mg per 100 μl of physiological solution. The fluorescent conjugate of the invention is excited and absorbs the light in the area from 570 nm to 790 nm and emits the light in the area from 680 to 850 nm, preferably at 850 nm.
The degree of substitution of the cypate residue of the formula II bound in the conjugate of hyaluronic acid or the salt thereof of the general formula I is from 0.1 to 2%, preferably 1.0%. The low degree of substitution of the cypate residue in the conjugate of the present invention is preferred, since it enables to the image distribution of the conjugate in vivo, without the structure of HA to be significantly modified.
The preparation of the conjugate itself lies in the synthesis of the fluorescent agent: Cypate, further in the activation of the carboxyl group of the fluorescent agent and following esterification of hyaluronic acid.
The activation of the carboxyl group of Cypate I of the formula III is performed using N,N′-carbonyl diimidalzole in the aprotic polar solvent (see Scheme 1, below) preferably selected from the group containing dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), formamide, or acetonitrile, more preferably DMSO. This activation is very effective even under the mild reaction conditions, wherein the reaction of the carboxyl group with N,N′-carbonyl diimidazole (CDI) results in the reactive intermediate mono-imidazolide (Cypate II of formula IV), where the reaction is driven by the release of carbon dioxide and imidazole (see Scheme 1, below). The activation reaction runs for 10 minutes to 24 hours, preferably 0.5 to 2 hours. The reaction temperature can be in the range from 20° C. to 60° C., preferably in the range from 22° C. to 25° C.
In the esterification of hyaluronic acid itself, shown in Scheme 2 (R+ is, as is defined above), is then imidazole eliminated from Cypate II from Scheme 1, and the ester bond between at least one hydroxyl group of hyaluronic acid and Cypate of formula IV is formed.
For the preparation of the conjugate, it is preferably used an acid form of hyaluronic acid or other organic salt for example tetrabutyl ammonium (TBA) (Scheme 2), that is used for the solubilization of hyaluronic acid in organic solvent (DMSO). Suitable molecule mass of the acid form or the other organic salt of hyaluronic acid for the given reaction is in the range from 5,000-250,000 g/mol, preferably (10,000-32,000 g/mol). Different molecule mass of HA of HA salt is not an obstacle for the reaction. The esterification of hydroxyl groups of HA in the aprotic polar solvent is further carried out by the addition of the fluorescent agent with the activated carboxyl group through CDI (N,N′-carbonyl diimidazole). The reaction runs at the presence of an organic base generated in situ in a form of imidazole or a added organic base selected for example from the group containing DABCO (1,4-diazabicyclo[2.2.2]octan), N,N,N′,N′-tetramethyl-1,6-hexanediamine, N-methyl morfoline, imidazole, triethylamine (TEA) or N,N′-diisopropylethylamine (DIPEA), preferably imidazole generated in situ and the polar aprotic solvent, as is defined above. The reaction of the conjugate forming is performed at the temperature from 40° C. to 80° C., preferably 40° C. to 60° C., more preferably 60° C. for 12 to 48 hours, preferably 24 hours A closer study revealed that the degree of substitution of HA with the fluorescent agent is dependent on an equivalent amount of Cypate I and also it is positively influenced by the presence of an equivalent of the organic base, the preferred combination is 0.5 molar equivalent of Cypate I:1 molar equivalent of HA:0.5 molar equivalent of N,N′-carbonyl diimidazole:0.5 to 3.5 molar equivalents of the organic base, more preferably 1 molar equivalent of the organic base. Thus, it applies that the molar ratio of Cypate I:hyaluronic acid or a salt thereof: N,N′-carbonyl diimidazole:organic base is 0.5:1:0.5:0.5 to 3.5 in the reaction mixture, preferably the molar ratio is 0.5:1:0.5:1. In the case of the organic base being generated in situ the molar ratio of cypate:hyaluronic acid or a salt thereof:N,N′-carbonyl diimidazole is 0.1:1:0.15 to 0.7:1:0.8, preferably 0.5:1:0.5.
The conjugate of hyaluronan with the heptamethine indocyanine fluorescent agent (or Cypate) of the general formula I, can be preferably further modified the forming of the hydrophobized fluorescent conjugate of the general formula I of the invention, where R+, R1 and n are, as is defined above, applying at the same time, that at least in one repeated unit at least one R1 is —C(═O)R2, wherein R2 is CxHy substituent, where x is an integer in the range of 5 to 17 and y is an integer in the range of 11 to 35, wherein it is a linear or branched, saturated or unsaturated C6-C18 aliphatic chain.
The degree of substitution of —C(═O)R2 in the conjugate of hyaluronic acid or the salt thereof of the general formula I is from 1 to 70%, preferably 5 to 12%. The hydrophobized fluorescent conjugate of the invention is excited and it absorbs the light in the range of wavelengths 570 nm to 790 nm and emits the light in the area of 680 to 850 nm, preferably at 850 nm.
The C6-C18 acyl chain is the chain of fat acids that is linked through the ester bond to at least one hydroxyl group of HA. It binds preferably to the primary hydroxyl group (see Scheme 3), that is generally suitable for the esterification. The fat acid can have the short (SCFA), middle (MCFA), or long (LCFA) aliphatic chain and it can be essential or nonessential. The activation of the fat acid of the general formula V
R2COOH (V),
wherein R2 is defined above,
is performed for example through the substituted or unsubstituted benzoyl chloride of the general formula VI
wherein R3 is one or more substituents selected from the group containing H, —NO2, —COOH, halogenides, C1-C6 alkyl alkoxy, preferably H;
in the presence of the organic base selected from the group containing e.g. DABCO (1,4-diazabicyclo[2.2.2]octan), N,N,N′,N′-tetramethyl-1,6-hexanediamine, N-methyl morfolin, triethylamine (TEA) or N,N′-diisopropylethylamine (DIPEA), preferably TEA. The example of the activation is shown in Scheme 3A. The reaction environment is made with the polar solvent selected from the group containing isopropyl alcohol (IPA), tetrahydrofuran (THF), preferably isopropyl alcohol, to form the reactive anhydride of the general formula VII
The esterification is performed with the activated carboxyl group of the fat acid in a mixture of water and the polar organic solvent miscible with water e.g. isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO) or tetrahydrofuran (THF), preferably isopropyl alcohol. The esterification is performed in the mixture of water and the polar solvent miscible with water, wherein the water amount is in the range from 50 to 80% v/v, preferably 50% v/v.
The reaction is also performed in the presence of the organic base preferably of amine selected from the group containing e.g. DABCO (1,4-diazabicyclo[2.2.2]octan), N,N,N′,N′-tetramethyl-1,6-hexanediamine, N-methylmorfoline, imidazole, triethylamine (TEA) or N,N′-diisopropyl ethylamine (DIPEA), more preferably triethylamine. In the method of preparation of the hydrophobized conjugate of the present invention, the activation of the fat acid of the general formula V is performed for 0.5 to 24 hours, at the temperature in the range 0° C. to 60° C., preferably 0.5 hours at the temperature 0° C. to 25° C. and the esterification of the fluorescent conjugate of hyaluronic acid or the salt thereof is performed for 0.5 to 2 hours, preferably 2 hours, at the room temperature, i.e. at the temperature in the range of 22° C. to 25° C.
According to a preferred embodiment of the method of preparation of the hydrophobized conjugate of the present invention the amount of organic base corresponds to 2 to 6 molar equivalents, preferably to 4 molar equivalents per dimer of hyaluronic acid or the salt thereof. The amount of the substituted or unsubstituted benzoyl chloride corresponds to 0.2 to 2.0 molar equivalents, preferably 0.6 molar equivalents per dimer of hyaluronic acid or the salt thereof. The amount of the fat acid corresponds to 0.2 to 2.0 molar equivalents, preferably 0.6 molar equivalents per the dimer of hyaluronic acid or the salt thereof. The closer study found out, that the degree of substitution of the hydrophobized conjugate of hyaluronic acid or the salt thereof of the invention with the fat acid depends on the equivalent amount of the activated fat acid and also it is positively influenced by the presence of the organic base.
The hydrophobized conjugate of hyaluronan with the heptamethine indocyanine fluorescent agent (Cypate) of the general formula I of the present invention can be preferably used for the encapsulation (noncovalent bond) of nonpolar agents, preferably drugs or nanoparticles with hydrophobic surface. The hydrophobized conjugate is able to aggregate and form systems similar to polymer micelles with their behavior. Thus a composition is formed on the basis of the aggregated hydrophobized fluorescent conjugate of the present invention that contains aggregates of the hydrophobized fluorescent conjugates and at least one or more nonpolar agents, preferably drugs, more preferably cytostatics, most preferably doxorubicin or paclitaxel, and/or nanoparticles, preferably superparamagnetic nanoparticles (i.e. spions). Spions are preferably on the basis of iron oxides (Fe2O3, Fe3O4), where the amount of iron in the composition is 0.3 to 3 wt. % preferably 1 to 1.5 wt. %. The size of superparamagnetic nanoparticles is 4 to 6 nm, preferably 5 nm. In the preferred embodiment, the composition contains the aggregated hydrophobized fluorescent conjugate of the present invention, wherein R1 —C(═O)C17H33 and nanoparticles, that are preferably superparamagnetic nanoparticles on the basis of iron oxides (Fe2O3, Fe3O4). Such composition can further preferably contain cytostatic, preferably doxorubicin or paclitaxel.
According to another embodiment of the present invention, the composition contains 2 to 15 wt. % of nonpolar agents in respect to the mass content of the hydrophobized fluorescent conjugate of hyaluronic acid or the salt thereof of present invention, preferably 2 to 6 wt. %.
The compositions of the present invention can be used in medicinal applications for in vivo imaging of tumors or for treating tumors.
In comparison with so far the described processes of preparation of the conjugates of hyaluronic acid with the cyanine fluorescent agents, the stated method of preparation of the conjugate of present invention brings about several advantages. In contrast to the technics described in the publications, it relates to the direct synthesis without using any linker or previous modification of hyaluronic acid, or fluorescent agent. During the activation occurs the release of imidazole and CO2 that are not toxic, and it can be easily removed from the reaction mixture. The release of imidazole can be further used in the preparation of the conjugate as in situ generated organic base and it need not be thus added as other organic base necessary for the reaction proceeding. The esterification of hyaluronic acid proceeds in the organic solvent e.g. in DMSO. The activator of cypate is CDI. CDI can be used for the conjugation of Cypate to HA without the necessity of isolation of the intermediate. The advantage in this case is that the selective modification of the primary hydroxyl of HA occurs even in the case of non-protected secondary hydroxyl groups of HA and no undesired side reaction like oxidation of HA in DMSO occurs (reaction Pfitzner-Moffatt).
Although the structure of Cypate contains two functional carboxyl groups, surprisingly no netting of the fluorescent conjugate of HA occurs (see
Similar ester derivatives of HA are not easy to obtain using other processes, or activators as are benzoyl chloride (BC) and 2,4,6-trichlorobenzoyl chloride (TBC) (WO2014082609). Neither ethyl chloroformiate can be used that can activate carboxyl group (WO 2012034544), as it leads to chromophore (cypate) degradation and loss of fluorescent properties. As another activator of carboxyl groups of hyaluronan in organic solvent is often used dicyclohexyl carbodiimide (DCC), its large disadvantage is, however, that it is indicated as strong allergen (Derm_Beruf. Umwelt. 1986; 34(4):110-1.) and it is highly toxic (Macrom. Rapid Commun. 2004, 25, 916-920).
The conjugate of hyaluronic acid with Cypate of general formula I and its hydrophobized conjugate of the present invention can be preferably excited in the area 570 nm to 790 nm and they emit at 680 to 850 nm, and thus suitable for the use in the medicinal applications for in vivo imaging of the conjugate distribution of the invention, preferably for in vivo imaging of organs selected from the group containing for example liver, skin; or imaging of tumors after the intravenous, intraperitoneal or subcutaneous administration.
These conjugates are able to penetrate after intravenous or intraperitoneal administration into tumor tissues (i.e. neoplasms), preferably into palpable tumors and/or into very small (non-palpable) tumors, and thus are suitable for the imaging to the diagnose disease, especially tumor disease. In comparison with the pentamethine compounds is heptamethine cyanine conjugate of hyaluronan advantageous in respect to fluorescent properties—especially of larger depth of the radiation penetration and further limitation of undesired autofluorescence. Diagnostics of tumor disease is preferably applicable for the tumor tissues selectively uptaking low-molecular hyaluronan (e.g. tissues with higher expression of CD44). The solubility of the hydrophobized conjugate of the present invention is 1 to 3 mg per 100 μl of the saline solution.
The hydrophobized conjugate of the present invention is in the term of the fluorescent properties very stable and after the intravenous administration it can be preferably imagined at least for 15 days without the necessity of the repeated administration of the conjugate. The conjugate of present invention is very easily concentrated even in small (palpably non-detectable) tumors. Another advantage of the hydrophobized conjugate of the present invention is the possibility of the noncovalent binding of the anticancer agent (cytostatic) and thus their usage for a construction of theranostics, i.e. carrier with diagnostic and therapeutic function at the same time. The main advantage of the hydrophobized conjugate of the present invention as a theranostic is the use of its ability to accumulate in tumor tissues (especially in breast tumors), long term imaging and application to treatment of tumor itself.
Cypate includes the structure of the general formula III (cypate I) or 1-[3-(2-carboxyethyl)-1,1-dimethyl-5,9b-dihydrobenzo[e]indol-3-ium-2-yl(chloride)]-octa-1,3,5,7-tetraenyl]-1,1-dimethyl-2H-benzo[e]indol-3-yl]propanoic acid, heptamethine indocyanine dye.
SS=degree of substitution=percentage amount of modified disaccharide units of hyaluronan per 100 disaccharide units of hyaluronan (100% states, that per 100 of disaccharide units of hyaluronan was detected 100 modifying units)
The term “room temperature” refers to the range of temperatures in the room of 22° C. to 25° C.
The equivalent (eqv.) refers to the dimer of hyaluronic acid, it refers to the molar equivalent if not stated otherwise.
The binding capacity is the amount of bound agent expressed in the percent by weight, if not stated otherwise.
The term “nonpolar agent” refers to a compound with symmetric distribution of charges. It refers to an agent that is soluble in organic solvents, especially in alcohols and insoluble in water.
NMR spectra were recorded on BRUKER AVANCE 500 at frequency 500.13 MHz (1H).
For the processing of experimental data was used the software by Bruker TOPSPIN 1.2 or software SpinWorks 3.1. For the interpretation of the spectra from NMR analyses were used abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet). For UV/Vis spectra measurement in the wavelength range 190-800 nm was used UV/Vis spectrophotometer Varian Cary 100. Fluorescent spectra were recorded on the apparatus PTI Quantarnaster 400. ESI-MS analyses of Cypate were performed on the mass spectrophotometer with ionic trap amaZon X (BrukerDaltonics) equipped with the electrospray ionizing source and the quadrupole mass analyzer. Measurements were performed in the positive and negative mode. The structure of conjugate of HA-cypate was confirmed using LC-MS analysis of the conjugate after its enzymatic cleaving with hyaluronan lyase. The mixture of oligosaccharides was separated on column Kinetex 1.7 um F5 100A (Phenomenex) using gradient 0.1% HCOOH in H2O and acetonitrile. The detection was performed on Synapt G2-Si in negative resolution mode with ionization with electrospray. Analysis of samples and molecule mass of initial hyaluronan was determined using the method SEC-MALLS (HPLC Alliance) with UV/VIS 2489 and the refractometry detector RID 2414 and the detector of light scattering mini DAWN TREOS. Data were processed using the software Astra Version 5.3.4.20 (Wyatt Technology EuropeGmbH). All in vivo imaging analyses were carried out on the apparatus IVIS Luminia XR Series III) on a laboratory mice of the strain Balb/c.
2.0 g (9.6 mmol) of 1,1,2-trimethyl-1H-benzindol and 2.2 g (14.3 mmol) 3-bromopropanoic acid were dissolved in 10 ml 1,2-dichlorobenzen and under the constant stirring warmed at 115° C. for 16 hours. The crude reaction mixture was cooled down to the room temperature and the resulting precipitate was washed with 1,2-dichloromethane (10×50 ml). The final product was separated using the filtration, dried under vacuum on the rotary evaporator (RE) and it was obtained in the form of light gray crystal powder (yield 2.2 g (64%)).
1H NMR (DMSO-d6, 500 MHz): δ 8.38 (d, J=8.35, 1Harom), 8.29 (d, J=9.00, 1Harom), 8.23 (d, J=8.35, 1Harom), 8.18 (d, J=9.00, 1Harom), 7.80-7.71 (m, 2Harom), 4.79 (t, J=6.95, 2H, —CH2—), 3.05 (t, J=6.95, 2H, —CH2—), 2.98 (s, 3H, —CH3), 1.76 (s, 6H, —CH3) ppm
ESI-MS: +MS [M]+=282
0.8 g (2.8 mmol) glutaconic dialdehyde dianiline hydrochloride was dissolved in 8 ml 1,2-dichloromethane and tempered to 0-5° C. 521 μl (5.5 mmol) of the anhydride of acetic acid, 481 μl (2.8 mmol) and DIPEA in the small volume of 1,2-dichlormethan (0.5 ml) was added dropwise into the solution and the reaction mixture was left while cooling and stirring to react for 3 hours. In the meantime 2 g of (5.5 mmol) 3-(2-carboxyethyl)-1,2,2-trimethyl-1H-benzo[e]indolium-bromide prepared in Example 1 and 0.9 g (11.0 mmol) of sodium acetate in the mixture of solvents acetonitrile/water 95/5 of volume 15 ml were brought to reflux and the first reaction mixture was added dropwise. The reaction proceeded for 18 hours under stirring at reflux in dark. The crude reaction mixture was cooled down to the room temperature, washed with 400 ml ethyl-acetate and 400 ml 1M HCl, the product was filtrated and dried under the low pressure. The resulting product was obtained in the form of dark green crystal powder yielding 1.62 g (88%).
1H NMR (DMSO-d6, 500 MHz): δ 8.25 (d, J=8.75, 2H), 8.07-7.97 (m, 6H), 7.82 (t, J=12.65, 1H), 7.73 (d, J=8.75, 2H), 7.69-7.62 (m, 2H), 7.54-7.48 (m, 2H), 6.60 (t, J=12.65, 2H), 6.47 (d, J=13.75, 2H), 4.43 (bt, 4H —CH2—), 2.77 (t, J=6.9, 4H —CH2—), 1.92 (s, 12H —CH3) ppm
ESI-MS: +MS [M]+=625; − MS [M-2H]−=623 m/z
UV/Vis: λabs.max=782 nm (MeOH)
87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N′-carbonyl diimidazole was added and under the constant stirring was let activate 2 hours at the room temperature. In the meantime 100 mg (0.27 mmol, 1 eqv.) acid form of hyaluronic acid Mw 14,000 g/mol was disolved in DMSO at 60° C. Then 15 μl (0.13 mmol, 0.5 eqv.) N,N′-methylmorfoline and the first reaction mixture without the previous isolation was added into the solution. The reaction proceeded under the constant stirring in dark at 60° C. 24 hours. The reaction was stopped by adding ten-fold of 100% isopropyl alcohol (AIPA) in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, when precipitation of the desired product occurred. The crude product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred into dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution of NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in the demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 89 mg (87%).
1H NMR (D2O) (
DOSY (D2O): (
SS=1.5% (determined from 1H NMR)
SEC-MALLS-LS-UV/Vis-RI: (
Fluorimeter: λem.max=695 nm (at λexc.=665 nm; H2O); (
Fluorescence of the conjugate excited with the laser wavelength λ=632.8 nm (
ESI-MS: [M-H]−=984 m/z (
87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N′-carbonyl diimidazole was added and under the constant stirring let to activate 30 min. at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw 14,000 g/mol in DMSO at 60° C. was added. The reaction proceeded under the constant stirring in dark at 60° C. for 24 hours. The reaction was stopped by addition of ten-fold volume of 100% isopropyl alcohol (AIPA) in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 89 mg (87%).
1H NMR (D2O) (
SS=1% (determined from 1H NMR)
Fluorimeter: λem.max=695 nm (at λexc.=665 nm; H2O)
17 mg (0.03 mmol, 0.1 eqv.) Cypate from Example 2 was dissolved in 1 ml DMSO, 6 mg (0.04 mmol, 0.15 eqv.) N,N′-carbonyl diimidazole was added and under the constant stirring was let to activate 30 min. at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw 14,000 g/mol in DMSO was dissolved at 60° C. The reaction proceeded under the constant stirring in dark at 60° C. 24 hours. The reaction was stopped with the addition of ten-fold volume of 100% isopropyl alcohol (AIPA) in respect to the initial reaction mixture volume and the saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution of NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in the demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 92 mg (90%).
1H NMR (D2O): cypate: δ 8.80 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, −5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.02 (12H, CH3, overlaid with signals of HA), HA: δ 4.62-4.39 (m, 2H anomer), 4.01-3.28 (m, 10H skeletal), 2.02 (s, 3H, —CH3) ppm
SS=0.7% (determined from 1H NMR)
Fluorimeter: λem,max=695 nm (at λexc.=665 nm; H2O)
122 mg (0.19 mmol, 0.7 eqv.) Cypate from Example 2 was dissolved in 1 ml DMSO, 34 mg (0.21 mmol, 0.8 eqv.) N,N′-carbonyl diimidazole was added and under the constant stirring was let to activate 30 min. at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw 14,000 g/mol in DMSO was dissolved at 60° C. The reaction proceeded under the constant stirring in dark at 60° C. 24 hours. The reaction was stopped with the addition of ten-fold volume of 100% isopropyl alcohol (AIPA) in respect to the initial reaction mixture volume and the saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution of NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in the demineralized water. The tube content was frozen and then lyophilized. The resulting product was obtain in the form of hyaluronan as green lyofilizate of mass 85 mg (88%).
1H NMR (D2O): cypate: δ 8.80 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, −5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.02 (12H, CH3, overlaid with signals of HA), HA: δ 4.62-4.39 (m, 2H anomer), 4.01-3.28 (m, 10H skeletal), 2.02 (s, 3H, —CH3) ppm
SS=1.3% (determined from 1H NMR)
Fluorimeter: λem.max=695 nm (at λexc.=665 nm; H2O)
87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) CDI was added and under the constant stirring was let to activate 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) the acid form of hyaluronic acid of Mw (14,000 g/mol) was dissolved in DMSO at 60° C. Then was 138 μl (0.79 mmol, 3 eqv.) DIPEA and the first reaction mixture added to this solution. The reaction proceeded under the constant stirring in dark at 60° C. 24 hours.
The reaction was stopped by the addition of ten-fold volume of AIPA in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then occurred the precipitation of the desired product. The product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred in the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution of NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as lyofilizate of mass 86 mg (84%).
1H NMR (D2O): cypate: δ 8.81 (s, 2H), 8.50-8.47 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.88-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, −5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH3, overlaid with signals of HA), HA: δ 4.62-4.38 (m, 2H anomer), 4.01-3.26 (m, 10H skeletal), 2.03 (s, 3H, —CH3) ppm
SS=1.5% (determined from 1H NMR)
SEC-MALLS-LS-UV/Vis-RI: (
Fluorimeter: λem.max=695 nm (at λexc.max=665 nm; H2O)
87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) CDI was added and under the constant stirring let activate for 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw (5.8×104 g/mol) in DMSO was dissolved at 60° C. Into this solution was then added 15 μl (0.13 mmol, 0.5 eqv.)N-methyl morfolin and the first reaction mixture without the previous purification. The reaction proceeded under the constant stirring in dark at 60° C. 24 hours.
The reaction was quenched by the addition of ten-fold of AIPA in respect to the initial volume of the reaction mixture and saturated solution of NaCl, then the precipitation of the desired product occurred. The crude product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as green lyofilizate of mass 95 mg (93%).
1H NMR (D2O): cypate: δ 9.2 (m, 2H), 8.82-8.77 (m, 2H), 8.46-8.43 (m, 2H), 8.29-8.19 (m, 2H), 8.16-8.10 (m, 2H), 8.08-7.89 (m, 2H), 7.89-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, −5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH3, overlaid with signals of HA), HA: δ 4.64-4.38 (m, 2H anomer), 4.04-3.21 (m, 10H skeletal), 2.03 (s, 3H, —CH3) ppm
[SS=1.0% (determined from 1H NMR)
SEC-MALLS-LS-UV/Vis-RI: (
Fluorimeter: λem.max=695 nm (at λexc.=665 nm; H2O))
87 mg (0.13 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N′-carbonyl diimidazole was added and with the constant stirring let activate for 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) acid form of hyaluronic acid of Mw (7.2×104 g/mol) in DMSO was dissolved at 60° C. 15 μl (0.13 mmol, 0.5 eqv.)N-methyl morfoline and the first reaction mixture was then added into this solution. The reaction proceeded with the constant stirring in dark at 60° C. 24 hours.
The reaction was stopped by addition of ten-fold volume of AIPA in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then occurred the precipitation of the desired product. The crude product was purified with 5×100 ml 100% AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as lyofilizate of mass 96 mg (94%).
1H NMR (D2O): cypate: δ 9.2 (m, 2H), 8.83-8.5 (m, 2H), 8.45-8.43 (m, 2H), 8.30-8.18 (m, 2H), 8.16-7.99 (m, 2H), 7.99-7.89 (m, 2H), 7.89-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.46 (m, 2H), 5.16, −5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH3, overlaid with signals of HA), HA: δ 4.64-4.38 (m, 2H anomer), 4.04-3.21 (m, 10H skeletal), 2.03 (s, 3H, —CH3) ppm
SS=1.0% (determined from 1H NMR)
SEC-MALLS-LS-UV/Vis-RI: (
Fluorimeter: λem.max=695 nm (at λexc.=665 nm; H2O))
87 mg (0.3 mmol, 0.5 eqv.) Cypate from Example 2 was dissolved in 2 ml DMSO, 22 mg (0.13 mmol, 0.5 eqv.) N,N′-carbonyl diimidazole was added and under the constant stirring let activate 2 h at the room temperature. 100 mg (0.27 mmol, 1 eqv.) of the acid form of hyaluronic acid of Mw (2.5×105 g/mol) was dissolved in DMSO at 40° C. N-methyl morfolin and first reaction mixture was then added 15 μl (0.13 mmol, 0.5 eqv.) into this solution. The reaction proceeded under the constant stirring in dark at 40° C. 24 hours
The reaction was stopped by the addition of ten-fold volume of AIPA in respect to the initial volume of the reaction mixture and the saturated solution of NaCl, then the precipitation of desired product occurred. The crude product was purified with 5×100 ml AIPA, dissolved in 50 ml demineralized water and transferred into the dialysis tube. The protonized form of HA was neutralized 1st day in 0.5% solution NaCl and 0.5% NaHCO3, further dialyzed 2nd and 3rd day in demineralized water. The tube content was frozen and lyofilized. The resulting product in the form of hyaluronan was obtained as lyofilizate of mass 93 mg (92%).
1H NMR (D2O): cypate: δ 9.2 (m, 2H), 8.83-8.5 (m, 2H), 8.45-8.43 (m, 2H), 8.30-8.17 (m, 2H), 8.16-7.99 (m, 2H), 7.99-7.89 (m, 2H), 7.89-7.81 (m, 2H), 7.79-7.73 (m, 2H), 7.58 (s, 2H), 7.51-7.42 (m, 2H), 5.16, −5.14 (m, 4H), 3.14-3.09 (m, 4H), 2.03 (12H, CH3, overlaid with signals of HA), HA: S 4.62-4.38 (m, 2H anomer), 4.01-3.26 (m, 10H skeletal), 2.03 (s, 3H, —CH3) ppm
SS=0.5% (determined from 1H NMR)
Fluorimeter: λem.max=695 nm (at λexc.=665 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, 382 μl (2.20 mmol, 3 eqv.) DIPEA and 4.5 mg (0.04 mmol, 0.05 eqv) DMAP were then added. 165 μl (1.32 mmol, 1.8 eqv.) hexanoic acid was dissolved in 2 ml AIPA, 255 μl (1.46 mmol, 2 eqv.) DIPEA 153 μl (1.32 mmol, 1.8 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at 0° C. After the period of time was everything quantitatively transferred to the first reaction mixture, then the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by addition of the high excess of AIPA and the saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3, 0.5% NaCl and further 8 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 230 mg (73%).
1H NMR additional signals compared with example 3 (D2O, 500 MHz): δ 2.4 (m, 2H, αCH2), 1.6 (m, 2H, βCH2), 1.31 (m, 4H, γCH2), 0.8 (m, 3H, —CH2—CH3) ppm
SS (acylation)=70% (determined from 1H NMR)
Fluorimeter: λem.max=700 nm (at λexc.=665 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water, and then was added 306 μl (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. In the meantime 94 mg (0.37 mmol, 0.5 eqv.) of hexadecanoic acid dissolved in 3 ml THF, 153 μl (1.10 mmol, 1.5 eqv.) TEA, 43 μl (0.37 mmol, 0.5 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.
The reaction was quenched by addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 189 mg (61%).
1H NMR additional signals to example 3 (D2O, 500 MHz): δ 2.76-2.66 (m, 2H, αCH2), 1.64-1.48 (m, 2H, βCH2), 1.31-1.08 (m, 24H, CH2), 0.98-0.78 (m, 3H, —CH2—CH3) ppm
SS acylation=7% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 am; H2O)
200 mg (0.49 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 10 ml demi water, and then 136 μl (0.98 mmol, 2 eqv.) TEA and 3 mg (0.02 mmol, 0.04 eqv.) DMAP were added. 38 mg (0.16 mmol, 0.3 eqv.) of palmitic acid dissolved in 3 ml THF, 68 μl (0.48 mmol, 1 eqv.) TEA, 17 μl (0.16 mmol, 0.3 ekv.) benzoyl chloride was added and let react with the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of high excess of AIPA and saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 76 mg (37%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 2.76-2.66 (m, 2H, αCH2), 1.64-1.48 (m, 2H, βCH2), 1.31-1.08 (m, 24H, CH2), 0.98-0.78 (m, 3H, —CH2—CH3) ppm
SS (acylation)=5% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 nm; H2O)
200 mg (0.49 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 10 ml demi water, and then 255 μl (1.47 mmol, 3 eqv.) DIPEA and 3 mg (0.02 mmol, 0.04 eqv.) DMAP were added. 38 mg (0.15 mmol, 0.3 eqv.) palmitic acid was dissolved in 3 ml THF, 85 μl (0.4-9 mmol, 1 eqv.) DIPEA, 17 μl (0.15 mmol, 0.3 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of high excess of AIPA and the saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 h against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 93 mg (45%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 2.76-2.66 (m, 2H, αCH2), 1.64-1.48 (m, 2H, βCH2), 1.31-1.08 (m, 24H, CH2), 0.98-0.78 (m, 3H, —CH2—CH3) ppm
SS (acylation)=5% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 nm; H2O)
300 mg HA-Cypate (0.73 mmol, 1 eqv.) from Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, 306 μl (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were then added. 186 μl (0.59 mmol, 0.8 eqv.) cis-octadec-9-enoid acid dissolved in 2 ml AIPA, 306 μl (2.20 mmol, 3 eqv.) TEA, 68 μl (0.59 mmol, 0.8 eqv.) benzoyl chloride was added and everything was let react under the constant stirring for 30 minutes at the room temperature. After the period of time was everything quantitatively transferred to the first reaction mixture, esterification then proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 210 mg (66%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2—CH3) ppm
SS (acylation)=10% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, then 306 μl (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 116 μl (0.36 mmol, 0.5 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 153 μl (1.08 mmol, 1.5 eqv.) TEA, 43 μl (0.36 mmol, 0.5 eqv.) benzoyl chloride was added and let react under the constant stirring for 30 minutes at the room temperature. After the period of time everything was quantitatively transferred to the first reaction mixture, the esterification then proceeded for 2 hours at the room temperature.
The reaction was quenched with the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilisate yielding 205 mg (73%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2—CH3) ppm
SS (acylation)=8% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate of HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 13 ml AIPA, 382 μl (2.20 mmol, 3 eqv.) DIPEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 139 μl (0.45 mmol, 0.6 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 229 μl (1.32 mmol, 1.8 eqv.) DIPEA, 51 μl (0.44 mmol, 0.6 eqv.) benzoyl chloride was added and let react under the constant stirring 30 minutes at 0° C. After the given period was everything quantitatively transferred to the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hrs in demineralized water. The product was obtained in the form of green lyofilizate yielding 204 mg (65%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2-CH3) ppm
SS (acylation)=12% (determined from 1H NMR)
Fluorimeter: λem.max.=710 nm (at λexc.=685 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, then was 306 μl (2.20 mmol, 3 eqv.) TEA and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP added. 231 μl (0.73 mmol, 1 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 204 μl (1.46 mmol, 2 eqv.) TEA, 85 μl (0.73 mmol, 1 eqv.) benzoyl chloride were added and let react with the constant stirring for 30 minutes at the room temperature. After the given period of time everything was quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in the demineralized water and transferred into dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 226 mg (70%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2-CH3) ppm
SS (acylation)=12% (determined from 11H NMR)
Fluorimeter: λem.max.=710 nm (at λexc.=685 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, then 375 μl (1.76 mmol, 2.4 eqv.) N,N,N′,N′-tetramethyl-1,6-hexanediamine and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP was added. 139 μl (0.44 mmol, 0.6 eqv.) of cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 375 μl (1.76 mmol, 2.4 eqv.) N,N,N′,N′-tetramethyl-1,6-hexanediamine, 51 μl (0.44 mmol, 0.6 eqv.) benzoyl chloride were added and let react under the constant stirring for 30 minutes at the room temperature. After the given period everything was quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hrs in demineralized water. The product was obtained in the form of green lyofilizate yielding 217 mg (70%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2—CH3) ppm
SS (acylation)=6% (determined from 1H NMR)
Fluorimeter: λem.max.=710 nm (at λexc.=685 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, and then 241 μl (2.20 mmol, 3 eqv.)N-methyl morfoline and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 139 μl (0.44 mmol, 0.6 eqv.) cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 193 μl (1.76 mmol, 2.4 eqv.)N-methyl morfoline, 51 μl (0.44 mmol, 0.6 eqv.) benzoyl chloride were added and let react under the constant stirring for 30 minutes at the room temperature. After the given period was everything quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of the product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further for 48 hrs in demineralized water. Product was obtained in the form of green lyofilizate yielding 264 mg (82%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2-CH3) ppm
SS (acylation)=12% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 nm; H2O)
300 mg (0.73 mmol, 1 eqv.) of the conjugate HA-Cypate prepared according to Example 3 was dissolved in 15 ml demi water and 11 ml AIPA, and then 246 mg (2.20 mmol, 3 eqv.) DABCO and 4.5 mg (0.04 mmol, 0.05 eqv.) DMAP were added. 139 μl (0.44 mmol, 0.6 eqv.) cis-octadec-9-enoic acid dissolved in 2 ml AIPA, 148 mg (1.32 mmol, 1.8 eqv.) DABCO, 51 μl (0.44 mmol, 0.6 eqv.) benzoyl chloride were added and let react with the constant stirring for 30 minutes at the room temperature. After the given period everything was quantitatively transferred into the first reaction mixture, and the esterification proceeded for 2 hours at the room temperature.
The reaction was quenched by the addition of the high excess of AIPA and saturated solution of NaCl, whereas the precipitation of product occurred. The crude product was washed with 4×200 ml AIPA, further dissolved in demineralized water and transferred into the dialysis tube. Dialysis proceeded for 24 hours against 0.5% NaHCO3 and 0.5% NaCl and further 48 hours in demineralized water. The product was obtained in the form of green lyofilizate yielding 183 mg (60%).
1H NMR (D2O, 500 MHz) additional signals to Example 3: δ 5.41-5.34 (m, 2H, CH═CH), 2.48-2.40 (m, 2H, —CH2—CO—), 1.68-1.54 (m, 2H, —CH2—CH2, —CO—), 1.40-1.23 (m, 24H, (—CH2)12), 0.91-0.68 (m, 3H, —CH2-CH3) ppm
SS (acylation)=3% (determined from 1H NMR)
Fluorimeter: λem.max=710 nm (at λexc.=685 nm; H2O)
150 mg of the acylated conjugate of HA-cypate prepared according to Example 16 was 2 hours dissolved in 15 ml demi water under the constant stirring on the magnetic stirrer. Then was gradually added 10 mg paclitaxel in 2 ml chloroform and the resulting mixture was evaporated (RE) to dry and then hydrated with demi water (15 ml). Unbound paclitaxel was removed with the filtration through 1.0 m glass filter and the resulting product was lyofilized.
The amount of unbound paclitaxel (HPLC determination): 4.2% (wt.)
150 mg of the acylated conjugate HA-cypate prepared according to Example 18 was 2 hours dissolved in 15 ml demi water with the constant stirring on the magnetic stirrer. Then 15 mg doxorubicin was gradually added in 2 ml chloroform and the resulting mixture was first sonicated (pulse sonication cca 15 min, 200 W, amplitude 65%, cycle 0.5 s) until reaching of the homogenous mixture and then evaporated (RE) to dry and then hydrated with demi water (15 ml). Unbound doxorubicin was removed with filtration through 1.0 μm glass filter and the resulting product was lyofilized.
The amount of unbound doxorubicin (HPLC determination): 7.5% (wt.)
150 mg of the acylated conjugate HA-cypate prepared according to Example 18 was 2 hours dissolved in 15 ml demineralized water with the constant stirring on the magnetic stirrer. Then 2 mg of spions on the basis of iron (Fe2O3, Fe3O4) (5 mm) and 20 mg doxorubicin was gradually added in 5 ml chloroform and the resulting mixture was first sonicated (pulse sonication, cca 15 min, 200 W, amplitude 65%, cycle 0.5 s) until reaching homogenous mixture and then evaporated (RE) to dry and then hydrated with demi water (15 ml). Unbound doxorubicin was removed with filtration through 1.0 m glass filter and the resulting product was lyofilized.
The amount of unbound doxorubicin (HPLC determination): 6.5% (wt.), unbound spions: 2% (wt.)
For in vivo experiments was HA-Cypate, prepared according to Example 3, dissolved in the saline solution (c=3.8 mg derivative in 100 μl solution) and then sterilized by filtration (0.22 μm). 50 μl (
HA-Cypate-C18:1 (SS=10%) prepared according to Example 15 was dissolved in phosphate buffer (c=3.6 mg of the conjugate in 100 μl solution and sterilized by filtration (0.22 μm). 100 μl of such prepared solution was applied intravenously to two model mice Balb/c (in narcosis) and observed its fluorescence in vivo for the period of 7 days (
HA-Cypate-C18:1 (SS=10%) prepared according to Example 15 was dissolved in the saline solution (c=1.8 mg of the conjugate in 100 μl solution) and sterilized by the filtration (0.22 μm). 100 μl of such prepared solution was applied intravenously to 3 model mice Balb/c (in narcosis) with non-palpable breast tumor (orthotopic administration 4T1 luc cells, displayable through chemiluminescence-detection of luciferase activity after i.p. injection of luciferin) and observable its fluorescence in vivo for period 72 hours (
To 9 model mice Balb/c (in narcosis) were orthotopically administered 4T1 luc cells, (cells displayable with chemiluminescence-detection of luciferase activity after i.p. application of luciferin) and breast tumor was let grow for 14 days. 14th day were mice divided into 3 groups (1 group=3 animals). To every group was 14th, 21th and 28th day intravenously applied 1 mg of selected conjugate in 100 μl phosphate buffer and then: to first group was applied HA-Cypate-C18:1 prepared in Example 15, to second group HA-Cypate-C18:l loaded with doxorubicin, prepared in Example 23 and to third group HA-Cypate-C18:1 loaded with doxorubicin and spions, prepared in Example 24. The radiation was evaluated for 35 days in vivo (
HA-Cypate-C18:l (SS=10%) prepared according to Example 15 was dissolved in the saline solution (c=0; 0.625; 1.25; 2.5 mg of the conjugate in 100 μl solution) and sterilized by the filtration (0.22 μm). 100 μl of such prepared solutions was applied intraperitoneally to 4 model mice Balb/C (in narcosis) with the breast tumor (35th day after orthotopic administration of 4T1 luc cells, displayable with chemiluminescence detection of luciferase activity after i.p. injection of luciferin) and observed its fluorescence in vivo for 7 days (
| Number | Date | Country | Kind |
|---|---|---|---|
| PV 2015-936 | Dec 2015 | CZ | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CZ2016/050048 | 12/22/2016 | WO | 00 |
