FLUORESCENT DYE IN TERNARY COMPLEX

Abstract

Pharmaceutical compositions and methods are presented for creating a ternary structure involving a fluorescent molecule, an intermediate carrier molecule, and a larger protein or polymer with a binding site receptive to the intermediate molecule or fluorescent/intermediate complex. The resulting ternary system improves the binding stability of the fluorescent dye to the protein, both in-vivo and in-vitro. This improved stability results in a longer half-life in medical use, enabling improved qualitative and quantitative use of the dye.

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Claims

1. A pharmaceutical composition of a ternary or three-separate molecules, in a specified range of molar ratios, that interact via non-covalent forces such as but not limited to H-bonds, π-stacking, hydrophobic interactions, salt-bridges, etc. in which the individual components are: a) a fluorescent dye, b) a saccharide with a high non-covalent affinity for a), and c) a suitable macromolecular carrier that can harbor a)+b).

2. The pharmaceutical composition of claim 1, wherein c) having the property of being either a component of the blood of a living being (e.g. serum albumin, plasma globulins) or a macromolecule capable of being tolerated in the blood of a living being (e.g. biodegradable polymers, liposomes or modified polypeptides).

3. The pharmaceutical composition of claim 1, wherein b) is a cyclodextrin.

4. The pharmaceutical composition of claim 1, wherein b) is a cyclodextrin which has been modified to enhance its binding affinity for c).

5. The pharmaceutical composition of claim 1, wherein c) is human serum albumin (HSA).

6. The pharmaceutical composition of claim 1, wherein a conjugating moiety such as modified N-hydroxysuccinimide or modified maleimide is used to tether b) to c).

7. The pharmaceutical composition of claim 4, wherein the composition comprises HSA in dimeric form or in a high molecular weight aggregates, such as nanoparticles.

8. The pharmaceutical composition of claim 1, wherein the fluorescent dye is ICG.

9. The pharmaceutical composition of claim 1, wherein the fluorescent dye is FLS.

10. The pharmaceutical composition of claim 1, wherein b) is Captisol.

11. The pharmaceutical composition of claim 1, wherein the molar ratios of a:b:c are 1:B:C, where B and C are chosen with the intent of ensuring that the percentage of a) that appears in the final product in the bound ternary state is close to 100%.

12. The pharmaceutical composition of claim 1, wherein a precise amount of the composition is provided in a single-use dispensing device.

13. The pharmaceutical composition of claim 1, lyophilized into a dried product for convenience of storage, transport, and usable life.

14. The pharmaceutical composition of claim 13, wherein the single-use dispensing device includes a mechanism for precise reconstitution of the lyophilized composition of before use.

15. A method for preparing the pharmaceutical composition of claim 1, using the fluorescent dye, saccharide, macromolecular carrier in a suitable molar ratio (such as 1:B:C, where C>B>1), and consisting of a sequential process of mixture, such as the steps of a. dissolving the saccharide in saline (or similar solvent), b. agitating the resulting solution, c. incubating the resulting solution, d. dissolving the fluorescent dye in solution and then adding it to the saccharide solution, e. agitating the resulting solution, f. incubating the resulting solution, g. adding a solution of the macromolecular carrier to the resulting solution, h. agitating the resulting solution, i. incubating the resulting solution, j. transferring to a suitable container, and k. storing under suitable light and temperature control.

16. The method of claim 15, wherein the addition of macromolecular carrier solution in step g) is performed with a large excess of fluorescent-saccharide complex relative to the macromolecular carrier, and before step j) a size-exclusion filter is used to remove unbound fluorescent-saccharide complex from the resulting product.

17. The method of claim 15, where the fluorescent dye is ICG.

18. The method of claim 15, where the saccharide is a cyclodextrin or modified cyclodextrin.

19. The method of claim 15, where the macromolecular carrier is HSA.

20. The method of claim 19, wherein in steps g) through i) the HSA protein structure is unfolded reversibly using temperature, pH or a chaotropic agent (e.g. ethanol or cholesterol) in order to enhance the inclusion of the fluorescent-saccharide complex inside the HSA.

21. The method of claim 15, wherein a conjugating moiety such as modified N-hydroxysuccinimide or modified maleimide is used to tether the fluorescent-saccharide complex to the macromolecular carrier.

22. The method of claim 15, where the final product of the method is lyophilized into a dried product for convenience of storage, transport, and usable life.

23. The method of claim 15, where the final product of the method is provided in a single-use dispensing device.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0050] FIG. 1A-1C: Structure of natural cyclodextrins, with α-CD, β-CD, and γ-CD shown respectively.

[0051] FIG. 1D: Conformational structure of β-CD. The larger opening of the bowl shape, on the right, is approximately 7.8 angstroms in inner diameter and 15.3 angstroms in outer diameter.

[0052] FIG. 2A: Tertiary Structure of HSA showing α-helical domains and drug binding sites.

[0053] FIG. 2B: Molecular simulation of β-CD binding to HSA.

[0054] FIG. 3A: Structure of ICG.

[0055] FIG. 3B: Structure of FLS.

[0056] FIG. 4A. Molecular simulation of ICG with relevant distances in angstroms.

[0057] FIG. 4B. Molecular simulation of ICG inserted into lipophilic cavity of β-CD.

[0058] FIG. 5A. Molecular simulation of β-CD-HSA.

[0059] FIG. 5B. Molecular simulation of ICG-β-CD-HSA.

[0060] FIG. 6A. Example of protein bound covalently to cyclodextrin through the use of N-hydroxysuccinimide.

[0061] FIG. 6B. Example of protein bound covalently to cyclodextrin through the use of modified maleimide.

DETAILED DESCRIPTION OF THE INVENTION

[0062] The ternary structure described above can be achieved by basic mixing, using the fluorescent, cyclodextrin, and HSA in a suitable molar ratio (such as 1:1:1), by introducing the components in an appropriate sequence under appropriate conditions. The following is one such procedure. One skilled in the art would recognize variations in this procedure that would also achieve the desired structure. [0063] a. 50 mg Captisol (Cydex's NC-04A-170167TS69) is dissolved in 1.0 ml Normal Saline, and [0064] b. subjected to vortex agitation for 3 minutes at full speed, and then [0065] c. incubated 15 minutes at normal room temperature. [0066] d. 25 mg Indocyanine Green (ICG, Cardiogreen Sigma 12633-100 mg) is rapidly dissolved in weigh boat with 0.3 ml distilled water pipetting up/down for 2 minutes and added rapidly to vial containing Captisol solution, and [0067] e. subjected to vortex agitation for 3 minutes at full speed, and then [0068] f. incubated 15 minutes at normal room temperature. [0069] g. 5 ml of 200 mg/ml Human Serum albumin, (Millipore-Sigma A3782—Fatty acid free, globulin free >99%) solution is added rapidly to the resulting solution of step f) and [0070] h. subjected to vortex agitation for 3 minutes at full speed, and then [0071] i. incubated 15 minutes at normal room temperature, [0072] j. transferred into sterile amber container, and [0073] k. Stored until use at 4-8° C.

[0074] In another embodiment, a large excess of HSA is used in step g), so that the molar ratios of fluorescent:CD:HSA are 1:1:N, where N>>1. This ensures that all HSA will be labelled. In this embodiment, in step j) the solution is sterile filtered through a size-exclusion filter such as a 0.2 um cellulose acetate syringe into the sterile amber container to remove excess ICG-CD complex that is unbound to HSA.

[0075] The product from step k) can be used directly or lyophilized into a dried product for convenience of storage, transport, and usable life.

[0076] For convenience in performance of indicator-dilution volume determinations, the product can be provided in precise quantities in a device capable of delivering the full quantity of the product, such as the Daxor Max-100 syringe.

[0077] Formation of the ternary complex can be confirmed and monitored by size-exclusion high-performance liquid chromatography (SEC-HPLC) coupled with a fluorescent detector. The ternary complex exhibiting the fluorescence eluting very close to the retention time of monomeric HSA. Stability of ICG fluorescence can be compared to free ICG in solution to verify increased performance.

[0078] The use of cyclodextrins in binary inclusion complexes to make drugs more soluble and modify their pharmacologic properties is widely known. A novel ternary inclusion system comprising A) the fluorescent probe inside B) the cyclodextrin and this inclusion complex inside C) Human Serum Albumin provides benefits from both known binary complexes: the stabilization and solubility benefits of CD-FP, and the preferential, stable binding of CD-HSA. The stable non-covalent of β-CD-HSA yields desirable properties for use in injection, particularly for quantitative measurements. The stability of FP-β-CD ensures that FP present in the system will be primarily in this bound state. The creation of the ternary complex ensures that FP will be stable and preferentially bound to HSA before injection. In addition the stoichiometric nature of the chemical specie will be known, this is worth noting since in many of the applications reported in the literature there is no certainty about the true composition of the chemical specie involved in the application, for instance mixtures of free FP and protein could exist or different loads of FP per protein can lead to ambiguous and non-reproducible results.

[0079] HSA starts denaturing reversibly for temperatures of up to 50° C. in a KCl 0.2 M buffer. The inclusion of ICG/CD could be achieved under a specific range of stirring and time, but the process can lead to aggregation if conditions are not controlled, i.e. above 65° C.—this phenomenon can be followed by SEC-HPLC. This is not necessarily a problem since HSA aggregates are non-toxic and have medical applications, e.g. perfusion scintigraphy with 99mTc-HSA. Changes in the 3D structure of HSA can be monitored via UV-vis absorption at 275 nm or circular dichroism.

[0080] HSA undergoes transformation and occurs in different isoforms (E: pH 2.6, F: pH 3.4, N: pH 5.6, B: pH 9.4, A). The molecule is stable from low pHs around 2 to 7. Between 7 and 9 a reversible unfolding occurs which can be helpful for non-covalent binding, however after pH of 10 there is a large change in the secondary and tertiary structure of HSA changes, causing its unfolding and an increase in the β-plated sheets, replacing α-helical structure that is generally irreversible (with degradation products such as fragments or aggregates that can be followed by SEC-H PLC).

[0081] HSA complexation can be facilitated with chaotropic agents. Concentration of ethanol below 40% v/v are recommended to avoid the formation of aggregates or fibrils. HSA can be reversibly unfolded using a 2-3 M solution of Guanidine HCl as long as the temperature is kept below 30° C.

[0082] The N-hydroxysuccinimide (NHS) group is a known conjugating agent to the lysine residue in proteins in general. Another option to couple small molecules to proteins is to take advantage of the maleimide reactivity, which targets cysteines residues specifically. HSA contains 35 cysteine residues, and all of them except one, Cys34 (in domain I), are involved in disulfide bonds stabilizing the structure of HSA; in this way this approach to conjugation can target a fixed location on the protein.