New forms of lutein and their uses

Abstract

Crystalline polymorphic lutein and cocrystal forms of lutein are disclosed. The new forms have enhanced bioavailability and stability, and may be used, for example, for topically delivering lutein to the eye for inhibiting or preventing cataracts, macular degeneration, and photooxidative damage to retinal pigment epithelium.

Description

NEW FORMS OF LUTEIN AND THEIR USESThis invention was made with Romanian Government support under contract number PN-III-P2-2.1-PED-2019-1288, within PNCDI III awarded by the Romanian Ministry of Education and Research, CCCDI - UEFISCDI. The Romanian Government has certain rights in the invention.The benefit of the 13 November 2023 filing date of United States provisional patent application serial number 63 / 598,201 is claimed under 35 U.S.C. § 119(e) in the United States, and is claimed under applicable treaties and conventions in all countries.TECHNICAL FIELD

[0001] This invention pertains to novel crystalline and cocrystal forms of lutein, for example to enhance delivery to tissues, and methods of making and using the novel forms of lutein, particularly for delivery of bioactive lutein to the eye, for example to prevent or treat disease in patients with age-related macular degeneration, cataracts, diabetic retinopathy, or photooxidative damage to retinal pigment epithelium.BACKGROUND ART

[0002] Lutein is a plant pigment, a xanthophyll, a dihydroxy carotenoid. The IUPAC name for lutein is p, E-carotene-3, 3'-diol. The structure of lutein is:

[0003] Because humans are not capable of synthesizing carotenoids in vivo, the lutein in human tissues is normally of dietary origin. Lutein is found, for example, in green plants (e.g., alfalfa, wheat grass, barley grass, kale, spinach, broccoli, green beans, green peas, lima beans, cabbage, collards, mustard greens, and turnip greens), certain flowers (e.g., marigold flower petals), certain yellow fruits and vegetables (e.g., carrots, peaches, mango, papaya, squash, and oranges), egg yolks, chicken skin, and chicken fat. In maize for example, lutein is found primarily in the horny endosperm. Marigold flower petals (Tagetes erecta) are also an excellent source of lutein, albeit more expensive than lutein derived from maize.

[0004] Lutein has a sequence of ten conjugated carbon-carbon double bonds. The conjugated structure allows lutein to function as a primary antioxidant in a biological system by scavenging radicals such as peroxyl radicals. However, the extensive conjugation also makes lutein susceptible to degradation by light, oxygen, and heat. The susceptibility to degradation makes it challenging to target the delivery of lutein to those tissues where it is most needed.

[0005] The hydroxyl groups make lutein more polar than its unmodified p-carotene analog. Lutein is soluble in both nonpolar and polar solvents. See Table 1 . able 1. Lutein: Physical Properties and Solubility in Various SolventsApril / May, pp. 64-67 (2001 )

[0006] Lutein can decrease the risk of certain diseases, and can reduce the symptoms of certain diseases, including diseases of the eye such as Age-Related Macular Degeneration (AMD). AMD is a degenerative condition of the region of the retina that is responsible for central vision. AMD is the most common cause of irreversible vision loss among older people. The carotenoids in the eye are concentrated in the inner retinal layer of the macula. Evidence from human studies suggests that dietary intake of carotenoids can lead to their accumulation in the retina, which is believed to provide protection against retinal degeneration. However, lutein is water-insoluble, making it difficult to effectively deliver bioactive lutein to target tissues, such as the retina, in a bioactive form without degradation. There is an unfilled need for improved methods and compositions to effectively deliver lutein to target tissues, such as the retina, in a living organism, in a bioactive form, without extensive degradation of the lutein.

[0007] Lutein also protects retinal pigment epithelial cells (RPE) from photo- oxidative damage through its ability to absorb short wavelength blue light, especially around 445 nm. Lutein can also modulate inflammation, and can help at least partially break the vicious cycle between oxidative stress and inflammatory response that can occur in RPEs. Furthermore, because lutein can quench singlet oxygen, lutein can help inhibit conditions resulting from oxidative stress. However, unmodified, naturally- occurring lutein has low water solubility, poor in vivo absorption, and low bioavailability.

[0008] Lutein’s presence in the eye, both in the lens and in the retina, and its known in vitro protective effect have led to a recommendation for dietary lutein supplementation for eye oxidative stress prevention. However, prior clinical studies have yielded mixed results for carotenoid oral supplementation for cataracts. Lutein, a hydrophobic molecule that is poorly soluble in water and that is itself susceptible to oxidation, is difficult to effectively deliver to target tissues in bioactive form withoutdegradation -- which is a possible reason for the mixed in vivo results that have been previously reported.

[0009] A topical bioadhesive formulation with lutein entrapped in poly(lactic-co- glycolic acid) (PLGA) nanoparticles (NPs) was disclosed by Sabliov et al. in WO / 2016 / 025394. See also Bodoki E., Vostinaru O., Samoila O., Dinte E., Bodoki A.E., Swetledge S., Astete C.E., Sabliov C.M., Topical nanodelivery system of lutein for the prevention of selenite-induced cataract, Nanomedicine. 2019 Jan;15(1 ):188-197.

[0010] There is an unfilled need for bioavailable formulations of lutein that are suitable for ocular administration, while retaining physical and chemical stability.

[0011] ON 111454187A discloses a crystalline form of lutein ethanol solvate characterized by X-ray diffraction peaks at 2.38, 3.88, 6.86, 10.9, 14.9, 15.3, 17.32,19.9, 20.5, 21 .08, 21 .64, 21 .68, 21 .98, 22.7, 24.36 °20 ± 0.2 °20.

[0012] CN 111548294A discloses a crystalline form of lutein methanol solvate characterized by X-ray diffraction peaks at 3.88, 6.01 , 8.07, 10.39, 12.67, 14.21 , 15.74, 16.54, 19.14, 21.27, 22.02, 23.66, 27.27 °20 ± 0.2 °20.

[0013] CN 113527165A discloses a crystalline form of lutein ester crystal form I characterized by X-ray diffraction peaks at 8.74, 11.23, 12.04, 12.58, 13.35, 15.95,17.10, 17.33, 18.08, 19.70, 23.26, 25.34, 27.23 °20 ± 0.2 °20; and a lutein ester crystal form II characterized by X-ray diffraction peaks at 11.36, 13.42, 14.66, 14.91 , 15.91 , 16.75, 18.79, 20.67, 23.07, 23.52, 24.65, 24.94, 25.81 °20 ± 0.2 °20.

[0014] Zhao, L etal., Encapsulation of lutein in liposomes using supercritical carbon dioxide, Food Research International 100, 168-179 (2017); and Chang, D et al, Improved oral bioavailability for Lutein by nanocrystal technology: formulation development, in vitro and in vivo evaluation, Artificial Cells, Nanomedicine and Biotechnology 46 (5), 1018-1024 (2017) disclose lutein polymorphs and a nanocrystal formulation of lutein said to improve oral bioavailability.

[0015] Gallic acid (Gal), 3,4,5-trihydroxybenzoic acid, is a phenolic acid with one aromatic ring, widely found in grapes, gallnuts, sumac, witch hazel, tea leaves, oak bark, and other plant sources. It is an antioxidant, reported to possess antiinflammatory activity, antitumor activity, antibacterial activity, and antifungal activity.

[0016] Gentisic acid (Gen), 2,5-dihydroxybenzoic acid, is an active metabolite of salicylic acid degradation. It has a broad spectrum of biological activity, such as antiinflammatory, antirheumatic, and antioxidant properties. It is a commonly-occurring aromatic acid in green plants.

[0017] 5-hydroxytryptophan (5-HTP) is a naturally-occurring aromatic amino acid, reported to have therapeutic activity against depression. It is slightly water-soluble, and has satisfactory bioavailability.

[0018] Kaur, P., Elsayed, A., Subramanian, J., Singh, A., Encapsulation of carotenoids with sucrose by cocrystallization: Physiochemical properties, characterization and thermal stability of pigments 2021, 140, 110810 discloses the use of a sucrose matrix to encapsulate heat-sensitive bioactive components such as carotenoids.

[0019] Bolla P.K., Gote V., Singh M., Patel M., Clark B.A., Renukuntla J., Lutein- Loaded, Biotin-Decorated Polymeric Nanoparticles Enhance Lutein Uptake in Retinal Cells, Pharmaceutics 2020, 12, 798 describes a pathway to improve lutein absorption in retinal cells with PLGA-PEG-biotin nanoparticles.

[0020] Lopez C., Meriadec C., David-Briand E., Dupont A., Bizien T., Artzner F., Anton M., Loading of lutein in egg-sphingomyelin vesicles as lipid carriers: thermotropic phase behaviour, structure of sphingosome membranes and lutein crystals, Food Research International 2020; 138, 109770 discloses the capacity of lipid carriers such as egg-sphingosomes to solubilize lutein inside bilayer vesicles.

[0021] Chang D., Ma Y., Cao G., Wang J., Zhang X., Feng J. & Wang W., Improved oral bioavailability for lutein by nanocrystal technology: formulation development, in vitro and in vivo evaluation, Artificial Cells, Nanomedicine and Biotechnology 2017; 46 (5), 1018-1024 discloses certain nanocrystal formulations of lutein.

[0022] Zheng, C„ Wang, H„ Xiao, Z„ Sun, Z„ Bao, J., Dai, W„ ... & Mei, X. (2024). Cocrystal of Lutein with Improved Stability and Bioavailability. ACS Omega. 9(34), 36389-36397 discloses the preparation of cocrystals of lutein and adipic acid.

[0023] Guo, W„ Du, S„ Xu, S„ Wang, Y„ Jia, L„ Liu, S„ ... & Wang, J. (2021 ). Unraveling the molecular mechanisms that influence the color and stability of four lutein crystal forms. Crystal Growth & Design, 21 (3), 1762-1777 discloses two polymorphs and two solvates of lutein.

[0024] There is an unfilled need for new forms of lutein to better take advantage of lutein’s antioxidant potential, and to improve its physicochemical stability during processing and storage. There is an unfilled need for new forms of lutein with enhanced stability (thermal, chemical, photo), improved dissolution rate, and improved bioavailability.SUMMARY OF THE INVENTION

[0025] We have discovered new crystalline and cocrystal forms of lutein. The new forms have enhanced bioavailability and stability (thermal, chemical, and photo-) as compared to unprocessed lutein. We have also discovered methods for preparing the new crystal and cocrystal forms. The novel compositions may be used, for example, for topically delivering lutein to target tissues such as the eye (including delivery of lutein to the retina) in bioactive form, while protecting the lutein from degradation. Preferably the compositions are admixed with a thermosensitive, bioadhesive gel to promote slow release of lutein or other antioxidant.

[0026] Lutein administered to the eye can be beneficial for such uses as inhibiting or preventing cataracts, macular degeneration, and photooxidative damage to retinal pigment epithelium. Preliminary results from a rat model are encouraging. Preliminary results showed that the novel compositions can successfully deliver lutein to the eye and deliver therapeutic benefit.

[0027] The present invention also provides cocrystals of lutein selected from the group consisting of lutein-gallic acid, lutein-gentisic acid, lutein-5-HTP and lutein-DL- a-Tocopherol; and their use, e.g., in drug administration for certain eye diseases.

[0028] As compared to what has been previously reported, the novel crystals and cocrystals are more stable, and they need not be prepared as solvates.DEFINITIONS

[0029] The term “polymorphs” refers to distinct solids that share the same molecular formula and the same primary molecular structure, and yet have distinct physical properties, for example a different crystal structure, or different inclusions of waters of hydration or other molecules of solvation. For example, hydrates are compounds containing water molecules in the crystal lattice, with a definite structure, and typically having a definite stoichiometric ratio of water. Solvated compounds more generally incorporate solvent molecules in the crystal structure, where the solvent molecules may include compounds other than water. With or without water or other solvent molecules, different crystalline forms of the same molecule can be formed by different methods of preparation. Different polymorphs of the same molecule can have different chemical and physical properties; and in particular different polymorphs candiffer in their bioavailability.

[0030] The term “cocrystal” refers to a solid, crystalline, single-phase material comprising two or more different molecular and / or ionic compounds, in a stoichiometric ratio, where the compounds are associated within the crystal lattice by nonionic or noncovalent bonds. While hydrates, other solvates, and salts are not excluded from this definition; neither is a composition considered to be a “cocrystal” merely by virtue of its being a hydrate, other solvate, or salt. The “coformer” is also part of a cocrystal composition. The “coformer” is a component that interacts ionically or nonionically in the crystal lattice with the other component, and that is not itself a solvent. Coformers are typically nonvolatile. In the context of the present disclosure, at least one of the components of the cocrystal is generally an active pharmaceutical ingredient (API), more specifically lutein. In the context of the present disclosure, the “coformer” is generally considered to be the non-lutein component of a cocrystal.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Fig. 1 depicts the observed X-ray powder diffraction pattern of lutein, new crystalline form (crystalline Lutein).

[0032] Figs. 2A and 2B depict, respectively, DSC and TGA-DSC thermograms of crystalline lutein, showing an endothermic event with an onset at 175.81 °C, corresponding to the melting point of this form of lutein.

[0033] Fig. 3. depicts a KF titration diagram of crystalline lutein, with 3.1 % water being detected.

[0034] Fig. 4. depicts a comparison of the X-ray powder diffraction patterns of crystalline lutein after physical stability assay at room temperature and 75 ± 5% relative humidity (RH), after 0, 2, and 4 weeks of exposure.

[0035] Fig. 5. depicts a comparison of the X-ray powder diffraction patterns of unprocessed lutein after physical stability assay at room temperature and 75 ± 5% relative humidity (RH), after 0, 2, and 4 weeks of exposure.

[0036] Fig. 6. depicts a comparison of the X-ray powder diffraction patterns of crystalline lutein after physical stability assay at 40°C and 75 ± 5% relative humidity (RH), after 0, 2, and 4 weeks of exposure.

[0037] Fig. 7. depicts a comparison of the X-ray powder diffraction patterns of unprocessed lutein after physical stability assay at 40°C and 75 ± 5% relative humidity (RH), after 0, 2, and 4 weeks of exposure.

[0038] Figure 8A depicts stability profiles of unprocessed lutein at room temperature (left bar of each pair), and at 40°C (right bar of each pair).

[0039] Figure 8B depicts stability profiles of crystalline lutein at room temperature (left bar of each pair), and at 40°C (right bar of each pair).

[0040] Figure 9 depicts in vitro diffusion profiles of lutein with different crystalline forms. From top to bottom, the six curves in the figure depict: lutein-gallic acid CC, lutein-5-HTP CC, lutein-gentisic acid CC, lutein-tocopherol CC, crystalline lutein, and unprocessed lutein.

[0041] Fig. 10 depicts the X-ray powder diffraction pattern of lutein-gallic acid cocrystal, Form 1.

[0042] Fig. 11 depicts a DSC thermogram of lutein-gallic acid cocrystal, Form 1 .

[0043] Fig. 12 depicts the X-ray powder diffraction pattern of lutein-gentisic acid cocrystal, Form 1.

[0044] Fig. 13 depicts the DSC thermogram of lutein-gentisic acid cocrystal, Forml .

[0045] Fig. 14 depicts the X-ray powder diffraction pattern of lutein-5-HTP cocrystal, Form 1.

[0046] Fig. 15 depicts the DSC thermogram of lutein-5-HTP cocrystal, Form 1 .

[0047] Fig. 16 depicts the X-ray powder diffraction pattern of lutein-DL-a-tocopherol cocrystal, Form 1.

[0048] Fig. 17 depicts the DSC thermogram of lutein-DL-a-tocopherol co-crystal, Form 1.

[0049] Fig. 18 depicts a comparison of the calculated PXRD patterns of: ethanol lutein solvate (AKEQAL, bottom curve, SC-XRD data from Guo et al. (2021); methanol lutein solvate (AKEGEF, middle curve, SC-XRD data from Guo et al. (2021)); and crystalline Lutein (top curve, this work, determined via ED analysis).

[0050] Fig. 19 depicts a comparison of the XRPD patterns of crystalline lutein as simulated from the ED data (bottom curve); and the actual recorded experimental data (top curve).MODES FOR PRACTICING THE INVENTIONExample 1 : A novel lutein polymorph

[0051] In one embodiment, the present invention provides a novel crystalline form of lutein (crystalline lutein) and its uses, for example in drug administration to patients with various eye diseases. The polymorph’s extended stability against degradation, even when not incorporated into nanoparticles, can circumvent potential concerns pertaining to possible toxicity or immunogenicity of engineered nanomaterials, or of residual organic solvents used in their synthesis.

[0052] This polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks, expressed in degrees 20 (± 0.2 °20), as described herein and in the Figures and Tables. One of skill in the art would be able to identify peaks of the lutein polymorph using the information set forth herein, including the XRPD peaks and other data.

[0053] Crystalline lutein of the present invention may be characterized by XRPD peaks at about 10.87, 11.28, 12.94, 14.12, 14.48, 14.90, 15.58, 16.12, 17.11 , 18.51 , 21 .49, 22.74, 24.19, and 24.32 °20 ± 0.2 °20. It may be further characterized by XRPD peaks at about 7.05, 9.78, 17.95, 19.79, 20.02, 26.09, 26.57, and 27.16 °20 ± 0.2 °20. The novel crystalline lutein polymorph may also be characterized by a differential scanning calorimetry (DSC) endotherm at about 181.12°C. It may also be characterized by an XRPD pattern and a DSC thermogram as depicted in Figures 1 and 2, respectively. Table 2 gives the d-spacing in A and the corresponding 20 values of crystalline lutein.Table 2. X-ray powder reflection peaks (up to 33° 20) and intensities (normalized) of crystalline lutein. The value 20 [°] represents the diffraction angle in degrees and the value d [A] represents the specified distance in A between the lattice planes. Particularly characteristic peaks for crystalline lutein in the X-ray powder diffraction pattern were found to be: d = (values in bold) ± 0.05 A.Example 2: A Method for producing the novel Lutein Polymorph

[0054] The novel crystalline form of lutein may be prepared, for example, by the following steps: a) dispersing lutein in an alcohol, e.g., methanol, ethanol, another alcohol, or a mixture of an alcohol and water; b) heating the mixture from step a) to a temperature of about 40°C for about 30 minutes; c) allowing the suspension from step b) to settle at 5°C; d) separating the solid phase from step c), and e) drying the separated solid phase from step d).This preparation procedure is straightforward to implement, and yet the resulting polymorph has novel and surprising properties. For example, the novel polymorph is stable, although it is not a solvate. Prior forms of lutein that have previously been reported have either been less stable, or they have incorporated solvent molecules, or both.Example 3: Alternative Method for producing the novel Lutein Polymorph

[0055] In an alternative embodiment, a novel crystalline form of lutein may be prepared by grinding / mechanochemical processing via the following steps: a) moistening solid lutein with a small volume of an organic solvent, for example an alcohol such as ethanol or methanol; b) subjecting the moistened mixture from step a) to a grinding treatment for about 30-60 minutes, such as grinding in a ball-mill (or other grinding apparatus known in the art), and c) harvesting the resulting crystalline form after drying at ambient conditions or under vacuum.This preparation procedure is also straightforward to implement. X-ray powder diffraction measurements support the conclusion that the polymorph produced by the procedure of this Example 3 is essentially the same as the polymorph produced by the procedure of Example 2. (Note: Minute volumes of solvent are often used inmechanochemical processing; such processing is still considered to present essentially solvent-free processing conditions.)

[0056] Each lutein polymorph will have distinct physical properties, and therefore can have distinct ocular biodistribution patterns. Preclinical efficiency has been assessed on animal (rodent) models, comparing the novel crystalline form of lutein to unprocessed lutein.Example 4: Slurry method for preparing the novel crystalline Lutein polymorph

[0057] 10 mg scale. Lutein (10 mg, 0.018 mmol) was dispersed in 3 mL methanol at room temperature (RT, 22-25°C). (Alternatively, another solvent such as ethanol or another alcohol may be used in place of methanol.) The mixture was heated to 40°C for 30 minutes. The resulting suspension was allowed to cool at RT (2 h) and to settle at 5°C overnight. The solids were then separated from the supernatant and left to dry under ambient conditions. After drying, the resulting material was analyzed by XRPD, which indicated the presence of a novel crystalline form of lutein.

[0058] 100 mg scale. Lutein (100 mg, 0.18 mmol) was dispersed in 12 mL methanol at room temperature (RT, 22-25°C). (Alternatively, another solvent such as ethanol or another alcohol may be used in place of methanol.) The mixture was heated to 40°C for 30 minutes. The resulting suspension was allowed to cool at RT (2 h) and to settle at 5°C overnight. The solids were then separated from the supernatant and left to dry under ambient conditions. After drying, the resulting material was analyzed by XRPD, which indicated the occurrence of the same novel crystalline form of lutein as that which had been prepared at the smaller, 10 mg scale.Example 5: Grinding method for preparing the novel crystalline Lutein polymorph

[0059] A novel crystalline Lutein polymorph, indicated by XRPD measurements to be the same novel polymorph as that resulting from the slurry technique of Example 4, was prepared by a grinding method.

[0060] 10 mg scale. Lutein (10 mg, 0.018 mmol) was moistened with 40 pLEthanol. Grinding was carried out at room temperature (RT) in one step of 30 min or 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials wereair-dried, harvested, and analyzed by XRPD, which indicated the presence of a novel crystalline form of lutein.

[0061] 50 mg scale. Lutein (50 mg, 0.088 mmol) was moistened with 100 - 200 pLEthanol. Grinding was carried out at room temperature (RT) in one step of 30 min or 60 min and 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were air-dried, harvested, and analyzed by XRPD, which indicated the presence of a novel crystalline form of lutein.Example 6: Physical and chemical stability assay

[0062] The physical stability of the crystalline lutein was studied under accelerated degradation conditions (RT and 40°C, 75 ± 5% RH) for up to four weeks. Under accelerated degradation conditions, the novel crystalline lutein had improved physical stability as compared to the unprocessed lutein form, as determined by XRPD analysis and crystallinity assessment. These assays followed generally the procedures of Chang, D et al, Nanomedicine and Biotechnology 46 (5), 1018-1024 (2017); and Zhao, L et al, Food Research International 100, 168-179 (2017). Physical stability results are summarized in Table 3 and are depicted in Figures 4-7.Table 3. Physical stability assayExample 7: In vitro diffusion test of lutein from a bioadhesive hydrogel

[0063] In vitro lutein permeability tests were carried out with different forms of lutein, using a bioadhesive hydrogel for topical ocular administration as otherwise described in Bodoki E., Vostinaru O., Samoila O., Dinte E., Bodoki A.E., Swetledge S., Astete C.E., Sabliov C.M., Topical nanodelivery system of lutein for the prevention of selenite-induced cataract, Nanomedicine. 2019 Jan;15(1 ):188-197. Experiments were conducted in duplicate, without exposure to natural light, using a Franz cell diffusion system (PermeGear, Bechenheim, Germany), and synthetic hydrophilic polyethersulfone (PES) membranes (pore size 0.45 pm, Sigma). The Franz cells had a diffusion surface of 0.6362 cm2, and held 10 mL receiving solution. 50mM TRIS buffer (pH = 7.1 ) admixed with 1 % (w / v) SDS was used as a receiving medium to confer sink conditions. The system was maintained at 37°C throughout the experiments in a thermostatic circulation bath (Julabo Corio C-B, Julabo GmbH, Seelbach, Germany), while the receiving solution was stirred continuously (500 rpm) with a magnetic stirrer. The synthetic membrane was positioned between the donor and receiver compartments of the diffusion cells, which had been maintained in the receiving solution for 30 minutes prior to equilibrate. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.027 g / mL. Then 100 pL aliquots of the receiving solution were taken at various times (up to 48 h), always replenishing with fresh receiving medium. Lutein concentration in the collected samples (expressed in ng / mL) was assessed by reverse-phase HPLC-UV (Figure 9).

[0064] As seen in Figure 9, all crystalline and co-crystalline forms of Lutein tested had a significantly improved diffusion profile in comparison with the unprocessed lutein (113 ng / mL after 48 h). A nearly 7-fold increase in lutein concentration was seen within the receiving compartment for the crystalline Lutein (783 ng / mL after 48 h) as compared to the unprocessed Lutein. Significantly higher levels of lutein were recorded for all the Lutein co-crystals tested, with up to 20-fold higher values in case of Lutein-Gallic acid CC (2368 ng / mL after 48 h).

[0065] The chemical stability profiles of the unprocessed and crystalline lutein appeared to correlate with their physical stability. Chemical stability was monitored during the degradation tests by reverse-phase HPLC-UV analysis. The lutein content in the samples collected at different times was determined from the recorded peakareas (Figure 8). Both unprocessed and crystalline lutein degraded more under the accelerated aging conditions (40°C). However the crystalline lutein degraded significantly more slowly, extending the shelf-life of lutein at RT by at least 2 weeks.Analytic MethodsX-ray powder diffraction (XRPD)

[0066] XRPD measurements were performed with an X-ray powder diffractometer Bruker D8 Advance instrument at room temperature, using a copper filled X-ray tube (40 kV x 40 mA) as the X-ray source, CuKa (A = 1 .5418 A), a 0.6 mm divergence slit, 2.5° Soller slits on both the primary and secondary beams, and a 1- dimensional LynxEye detector (aperture angle 2.91593°). Data were collected in the range 3.8-33° 20 at 0.02° increment steps, and a scan speed of 0.3 s / step.Differential Scanning Calorimetry (DSC)

[0067] Differential scanning calorimetry (DSC) was performed with a Shimadzu DSC-60 calorimeter in the range 20-300°C, with a heating rate of 10°C min-1, using a crimped aluminum sample cell with 60 mL / min nitrogen flow.Karl Fischer titration (KF)

[0068] Moisture levels were assessed using the coulometric Karl Fischer method for determination of low water content. The measurement was made at room temperature with a Titration TitroLine® 7500 KF trace apparatus with double platinum electrode.High-performance liquid chromatography (HPLC) with UV detection

[0069] Liquid chromatographic assays were conducted on an Agilent series 1200 LC system equipped with a DAD detector. Lutein was separated on a C8- based reverse phase column (4.6 x 50 mm, 5 pm) at 40°C, using a mixture of 80% ACN and 20% formic acid 0.1 % as the mobile phase. The injection volume was 50 pL, and the detection wavelength was 446 nm. The isocratic flow rate was 0.8 mL / min.Animal studiesExample 8. Chemically-induced cataracts in rat pups

[0070] Fourteen pregnant Wistar female albino rats were obtained from the Laboratory Animal Facility of the luliu Hatieganu University of Medicine and Pharmacy in Cluj-Napoca, Romania. Each female rat and litter of pups were housed in plastic cages on a 12 h lighting cycle at constant temperature (22°C) with free access to rat chow and tap water. The pups from each female rat were randomized into seven groups as follows:

[0071] Group 1 (selenite group, control): no exposure to lutein.

[0072] Group 2 (unprocessed Lutein): treated topically, once a day, with a corneal application (1 drop in each eye) of unprocessed lutein in the bioadhesive hydrogel. 4.50 mg unprocessed lutein were dispersed into 3.498 mL of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1 .035 g / mL. The final concentration of lutein was -1280 pg I ml. (The intention was that each of the animals in the treatment Groups would receive approximately the same amount of lutein.)

[0073] Group 3 (crystalline Lutein): treated topically, once a day, with a corneal application (1 drop in each eye) of crystalline lutein in the bioadhesive hydrogel. 4.50 mg crystalline lutein were dispersed into 3.498 mL of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1 .035 g / mL. The final concentration of lutein was -1280 pg / ml.

[0074] Group 4 (Lutein-Gallic acid CC): treated topically, once a day, with a corneal application (1 drop in each eye) with -1280 pg I mL (from the lutein-gallic acid cocrystal example described below) in the bioadhesive hydrogel. 1.66 mg of lutein-gallic acid CC were dispersed in 1.000 ml of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.035 g / mL.

[0075] Group 5 (Lutein-Gentisic acid CC): treated topically, once a day, with a corneal application (1 drop in each eye) with -1280 pg lutein I mL (from the lutein-gentisic acid cocrystal example described below) in the bioadhesive hydrogel. 1.62 mg of lutein-gentisic acid CC were dispersed in 1.000 ml of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1 .035 g / mL.

[0076] Group 6 (Lutein-5-HTP CC): treated topically, once a day, with a corneal application (1 drop in each eye) with -1280 pg lutein I mL (from lutein-5HTP cocrystal example described below) in the bioadhesive hydrogel. 1 .77 mg of lutein- 5-HTP CC were dispersed in 1 .000 ml of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1 .035 g / mL.

[0077] Group 7 (Lutein-Tocopherol CC): treated topically, once a day, with a corneal application (1 drop in each eye) with -1280 pg lutein I mL (from lutein- tocopherol cocrystal example described below) in the bioadhesive hydrogel. 2.25 mg of lutein-tocopherol CC were dispersed in 1.000 ml of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1 .035 g / mL.

[0078] Selenite-induced cataract in the rat is a rapid and convenient model for nuclear cataracts. Administering selenite to suckling rat pups induces cataracts. On day 13 post-partum, cataracts were induced in all Group 1-7 animals with a single, intraperitoneal injection of sodium selenite (Na2SeOs), 30 pmol / kg. Subsequently, the animals from Groups 2-7 were treated daily, in accordance with the protocols described above. On day 21 post-partum, cataract development was evaluated by slit-lamp examination (Table 4). Eyes were scored into one of five stages: stage 0 (no cataracts), stage 1 (slight nucleus opacity, occupying less than 25% of the diameter of the nucleus), stage 2 (mild nucleus opacity, a central white opacity occupying less than half the diameter of the nucleus), stage 3 (dense opacity, a central while opacity occupying between 50% and 75% of the nucleus) and stage 4 (dense, white opacity over more than 75% of the nucleus). Statistics were performed in SPSS 14.0 for Windows, and Excel. The variables were checked for normal distribution with the Shapiro-Wilk test. Groups were compared with the Wilcoxon test. Statistical significance was set at p < 0.05.

[0079] The experimental protocol was approved by the Ethics Commission of the luliu Hatieganu University of Medicine and Pharmacy and the National Veterinary Authority (Authorization no. 264 / 25.06.2021 ), with experiments conducted in accordance with EC Directive 86 / 609 EEC regulating the use of laboratory animals for scientific experiments.Table 4: Observed distribution of cataract severity in rat pups, with cataracts induced by selenite 13 days postpartum

[0080] According to the Saphiro Wilk test, none of the data sets were normally distributed. Therefore the Wilcoxon statistical test was applied. The treatments with unprocessed lutein (Group 2) and with crystalline lutein (Group 3) both showed some reduction in severity of developed cataracts, with statistically significant differences (p<0.05) from control (Group 1 ). A nearly 70% reduction in mean cataract severity as compared to control was seen for the topical treatment with the hydrogel incorporating crystalline lutein. A significant, but smaller reduction in cataract severity (-33%) was seen for the topical treatment with the unprocessed Lutein formulation (Group 2).

[0081] Of the topical treatments with lutein cocrystals, all but one (lutein- tocopherol CC, Group 7) demonstrated a statistically significant reduction (-54- 57%, p<0.05) in cataract severity as compared to control (Group 1 ). The treatments with gallic acid, gentisic acid, and 5-HTP cocrystals (Groups 4-6) had better outcomes in mean cataract severity as compared with unprocessed lutein (Group 2), but not as good as that for the crystalline lutein (Group 3).Example 9. Streptozotocin-induced diabetic retinopathy in adult rats

[0082] Three-month old Wistar rats were obtained from the Practical Skills and Experimental Medicine Centre of the luliu Hatieganu University of Medicine and Pharmacy in Cluj-Napoca, Romania. The animals were housed in polycarbonate cages on a 12 h light / dark cycle, with free access to standard rat chow and tap water. Diabetes mellitus (DM) was induced by a single intraperitoneal injection of streptozotocin (STZ, 65 mg / kg) (Sigma-Aldrich, USA). After 72 hours glucose levels were determined in blood drawn from the tail vein using a digital AccuChek glucometer (Roche Diagnostics, Germany). Excluded from the study were rats with unconsolidated diabetes (blood glucose <250 mg / dL). All rats with a fasting blood glucose level over 250 mg / dL were considered diabetic, and the diabetic rats were randomly assigned to one of the study groups:

[0083] Group 1 (diabetic, control): no treatment.

[0084] Group 2 (diabetic, treated with crystalline lutein) was treated topically, once a day, with a corneal application (1 drop in each eye) of crystalline Lutein in bioadhesive hydrogel. 4.71 mg crystalline lutein were finely dispersed into 3.66 ml of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.027 g / mL. The final concentration of lutein was -1280 pg / mL. (The intention was that each of the animals in the treated Groups would receive approximately the same concentration of lutein.)

[0085] Group 3 (diabetic, lutein-gallic acid CC) was treated topically, once a day, with a corneal application (1 drop in each eye) of lutein-gallic acid CC in bioadhesive hydrogel. 5.06 mg lutein-gallic acid CC were finely dispersed into 3.04 mL of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.027 g / mL. The final concentration, expressed as lutein equivalent, was -1280 pg / mL.

[0086] Over a period of 9 weeks, the animals from groups 2 and 3 were treated daily, in accordance with the protocols described above. The animals’ blood glucose and weight were monitored throughout the experiment (namely, at weeks 3, 6 and 9). After 9 weeks, the animals were anesthetized by an intraperitoneal injection of a 0.1 mL / 100 g ketamine-xylazine cocktail before an OCT investigation to evaluate the progress of diabetic retinopathy (See Table 5).

[0087] Cataract development was also evaluated by slit-lamp examination. Mydriasis was not needed. Cataracts were scored into one of five stages as described. Morphology of the cataracts was noted, together with the presence of any nuclear or cortical opacities. All examined eyes were photographed.

[0088] Retinas were examined with a Fundus camera (7 M pixels) and Ocular Coherence Tomography (SD-OCT). Retinal structure was observed with OCT, and retinal thickness maps were prepared. (OCT examination was not feasible in eyes with denser cataracts.) The eyes were aligned so the optic nerve lay in the center of the image, to facilitate comparisons between images of different retinas. SD- OCT produced three thickness maps centered on the optic nerve, each having a diameter of 3.5 mm: total retinal thickness, internal retinal thickness, and external retinal thickness. The demarcation between internal and external retina lies at the junction between the internal plexiform and the internal nuclear layers. Retinal photoreceptors were investigated in the external retinal layer. Each map was subdivided into 4 quadrants: superior, inferior, nasal, and temporal.

[0089] At the end of the experiment all rats were sacrificed by cervical dislocation under general anesthesia. Then the retina, sclera, and crystalline lens were surgically collected from each individual for histopathological and biochemical analyses.

[0090] Statistics were performed in SPSS 14.0 for Windows and Excel. The variables were checked for normal distribution with the Shapiro-Wilk test. Groups were compared by the ANOVA and one-sample t-test. Statistical significance was set at p < 0.05.

[0091] The experimental protocol was approved by the Ethics Commission of the luliu Hatieganu University of Medicine and Pharmacy and the National Veterinary Authority (Authorization no. 264 / 25.06.2021 ), the experiment being conducted in accordance with EC Directive 86 / 609 EEC regulating the use of laboratory animals for scientific experiments.Example 10, ResultsTable 5: Outer and total retinal thickness 9 weeks after induction of diabetes in adult rats‘(statistically significant difference from Group 1 , p<0.05)

[0092] According to the ANOVA test, Group 2 (crystalline lutein) and Group 3 (lutein-gallic acid CC) both showed statistically significant differences (p<0.05) from control (Group 1 ). There was a significant reduction in retinal thickness in the controls in the absence of treatment, especially in the outer retina. Lutein treatment was effective in helping to preserve photoreceptor cells.

[0093] Diabetic rats tended to develop cortical cataracts. 71% of controls showed various levels of cataract formation, while 29% of controls had no cataracts. In the treatment groups, 54% in Group 2 and 62% in Group 3 developed cataracts (Table 6).Table 6: Observed distribution of cataract severity 9 weeks after the induction of diabetes in adult ratsCataract stageCataract occurrenceAnimal group Mean ± SDN Stage Stage Stage Stage Stage 0 1 2 3 41: Positive control(no treatment)1.18+1.43*2: Crystalline Lutein 22 10 5 3 1 3 (p=0.005)1 .29+1 .37*: Lutein-gallic acid CC 24 9 6 5 1(p=0.006)‘(statistically significant difference from Group 1 , p<0.05)

[0094] Both forms of Lutein used in this experiment (viz., crystalline lutein and lutein-gallic acid CC) were effective in reducing cataract formation. According to the single-sample t-test, both treatment groups showed statistically significant differences (p<0.05) from control (Group 1 ), with a 45% reduction in mean cataract severity for crystalline lutein, and a 40% reduction for lutein-gallic acid CC.Example 11. Chemically-induced cataracts in rat pups

[0095] Eight pregnant Wistar female albino rats were obtained from the Laboratory Animal Facility of the luliu Hatieganu University of Medicine and Pharmacy in Cluj-Napoca, Romania. Each female rat and litter of pups were housed in plastic cages on a 12 h lighting cycle, at constant temperature (22°C) with free access to rat chow and tap water. The pups from each female rat were randomized into five groups as follows:

[0096] Group 1 (selenite group, control): no exposure to lutein.

[0097] Group 2 (empty PLGA NPs, no lutein): treated topically, once a day, with a corneal application (1 drop in each eye) of empty PLGA NPs (poly(lactic-co- glycolic) acid nanoparticles) in bioadhesive hydrogel. 478.6 mg empty PLGA NP (lyophilized product) were dispersed into 9.727 mL of bioadhesive hydrogel, to have the same concentration of solid material per mL of hydrogel (49.2% w / v) as for the lutein-loaded PLGA NPs of Group 3. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.031 g / mL. The final concentration of lutein was 0.

[0098] Group 3 (crystalline lutein incorporated into PLGA NPs): treated topically, once a day, with a corneal application (1 drop in each eye) of crystalline lutein encapsulated into PLGA-NPs in bioadhesive hydrogel, as otherwise described in WO 2016 / 025394. The equivalent of 8.29 mg of lutein was dispersed into 6.472 mL of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.031 g / mL. The final concentration of Lutein was -1280 pg / mL. (The intention was that each of the animals in the treatment groups should receive approximately the same concentration of lutein.)

[0099] Group 4 (lutein-gallic acid CC incorporated into PLGA NPs): treated topically, once a day, by corneal application (1 drop in each eye) with -1280 pg lutein / mL from lutein-gallic acid CC encapsulated into PLGA NPs in bioadhesive hydrogel, as otherwise described in WO 2016 / 025394. The equivalent of 7.29 mg of lutein were dispersed in 5.692 ml of bioadhesive hydrogel. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.031g / mL. The final concentration of lutein was -1280 pg / mL.

[0100] Group 5 (plain hydrogel, no PLGA NPs, no lutein): treated topically, once a day, by corneal application (1 drop in each eye) with bioadhesive hydrogel and no lutein. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1 .031 g / mL. The final concentration of Lutein was 0.

[0101] On day 13 post-partum, cataracts were induced in all animals in Groups 1-5 with a single, intraperitoneal injection of sodium selenite (Na2SeOs), 30 pmol / kg. Subsequently, the animals from groups 2-5 were treated daily, in accordance with the protocols described above. On day 21 post-partum, cataract development was evaluated by slit-lamp examination (Table 7). Cataracts were scored into one of five stages, as otherwise described above. Statistics were performed in SPSS 14.0 for Windows, and Excel. The variables were checked for normal distribution with the Shapiro-Wilk test. Groups were compared with the Wilcoxon test. Statistical significance was set at p < 0.05.

[0102] The experimental protocol was approved by the Ethics Commission of the luliu Hatieganu University of Medicine and Pharmacy and the National Veterinary Authority (Authorization no. 264 / 25.06.2021 ), the experiment being conducted in accordance with EC Directive 86 / 609 EEC regulating the use of laboratory animals for scientific experiments.Table 7: Observed distribution of cataract severity in rat pups having cataracts induced 13 days postpartum‘(statistically significant difference from Group 1 , p<0.05)

[0103] According to the Saphiro Wilk test, none of the data sets were normally distributed. Therefore, the Wilcoxon statistical test was applied. The treatments with crystalline lutein (Group 3) and with lutein-gallic acid CC (Group 4) incorporated into PLGA NPs both reduced mean cataract severity, by 27% and 41 %, respectively, with statistically significant differences (p<0.05) from the control (Group 1 ).

[0104] The topical treatment with crystalline lutein, and the treatment with luteingallic acid CC incorporated into the polymeric nanocarrier were less effective in reducing cataract severity than were the same forms of lutein directly dispersed into the hydrogel, lower by 42% and 15% respectively. These results demonstrated the significant difference that the crystalline form of lutein has on bioactivity and effectiveness. On the other hand, lutein-gallic acid CC encapsulated into PLGA NPs (Group 4) performed slightly better (by -14%) than crystalline lutein encapsulated into the polymeric nanodelivery system (Group 3), perhaps due to the additional antioxidative effect of the coformer molecule. No change in cataractprogression was observed with any topical treatment in the absence of lutein - namely empty PLGA NPs (Group 2) or plain hydrogel (Group 5), as compared to the non-treated control (Group 1 ).

[0105] We have discovered several lutein cocrystals that do not appear to have been previously reported. Our initial attempts to prepare cocrystals of lutein with a large number of potential coformer compounds had been largely unsuccessful. Finding successful coformer compounds from the potential candidates is not trivial. We eventually discovered several compounds that worked as successful coformers with lutein. Particularly effective coformers with lutein included: gallic acid, gentisic acid, 5- HTP, and DL-a-Tocopherol. The percentage recovery of lutein in these 1 :1 cocrystals, as assayed by HPLC-UV analysis were: 92.45% (Gallic acid), 94.81% (Gentisic acid), 88.18% (5-HTP) and 102.91% (DL-a-Tocopherol).

[0106] To identify this handful of successful coformers, we tested a much larger group of potential compounds -- most of which did not successfully produce co-crystals with lutein. We have tested at least twenty-eight such unsuccessful potential coformer candidates, including compounds such as L-histidine, caffeine, mannitol, urea, salicylic acid, and camphor-10-sulfonic acid.Example 12

[0107] Lutein-Gallic acid cocrystal, Form 1 is characterized by XRPD peaks at about 6.46, 10.08, 17.25, and 19.15 °20 ± 0.2 °20. It may be further characterized by XRPD peaks at about 7.20, 13.64, 14.31 , 15.63, 16.19, 20.24, 21.49, 24.36, and 25.95 °20 ± 0.2 °20. Lutein-Gallic acid cocrystal, Form 1 may also be characterized by a DSC endotherm at about 153.93°. The lutein and the gallic acid are preferably in a 1 :1 stoichiometric ratio. This cocrystal may also be characterized by an XRPD pattern and DSC thermogram as depicted in Figures 10 and 11 , respectively. Table 8 gives the d- spacing in A and the corresponding 20 values of lutein-gallic acid cocrystal, Form 1 .Table 8. X-ray powder reflections (up to 33° 20) and intensities (normalized) for luteingallic acid cocrystal, Form 1. The value 20 [°] represents the diffraction angle in degrees, and the value d [A] represents the specified distances in A between the lattice planes. Particularly characteristic peaks for lutein-gallic acid cocrystal, Form 1 , in the X-ray powder diffraction pattern have the values shown in bold (±0.05A).Example 13

[0108] Lutein-Gentisic acid cocrystal, Form 1 is characterized by XRPD peaks at about 10.95, 15.71 , 16.15, 17.51 , and 24.80 °20 ± 0.2 °20. It may be further characterized by XRPD peaks at about 14.34, 14.64, 15.01 , 17.25, 18.65, 21.60, and 24.40 °20 ± 0.2 °20. Lutein-gentisic acid cocrystal, Form 1 may also be characterized by a DSC endotherm at about 139.24°. The lutein and the gentisic acid are preferably in a 1 :1 stoichiometric ratio. This cocrystal may also be characterized by an XRPD pattern and DSC thermogram such as depicted in Figures 12 and 13, respectively. Table 9 gives the d-spacing in A and the corresponding 20 values for Lutein-Gentisic acid cocrystal, Form 1 .Table 9: X-ray powder reflections (up to 33° 20) and intensities (normalized) for lutein- gentisic acid cocrystal, Form 1 . The value 20 [°] represents the diffraction angle in degrees, and the value d [A] represents the specified distances in A between the lattice planes. Particularly characteristic peaks for lutein-gentisic acid cocrystal, Form 1 , in the X-ray powder diffraction pattern have the values shown in bold (±0.05A).Example 14

[0109] Lutein-5-HTP cocrystal, Form 1 is characterized by XRPD peaks at about 11.08, 14.22, 16.41 , 17.49, 21.04, 23.62, 25.32, 25.70, and 28.20 °20 ± 0.2 °20. It may be further characterized by XRPD peaks at about 14.59, 14.96, 15.66, 16.18, 17.26, 18.52, 19.17, 21.56, 22.77, and 24.36 °20 ± 0.2 °20. Lutein-5-HTP cocrystal, Form 1 may also be characterized by DSC endotherms at about 59.32°C and 173.14°C, and a DSC exotherm at about 104°C. The lutein and the 5-HTP are preferably in a 1 :1 stoichiometric ratio. This cocrystal may also be characterized by an XRPD pattern and DSC thermogram such as depicted in Figures 14 and 15, respectively. Table 10 provides the d-spacing in A and the corresponding 20 values of Lutein-5-HTP cocrystal, Form 1.Table 10: X-ray powder reflections (up to 33° 20) and intensities (normalized) of lutein- 5-HTP cocrystal, Form 1 . The value 20 [°] represents the diffraction angle in degrees and the value d [A] represents the specified distances in A between the lattice planes. Particularly characteristic peaks for lutein-5-HTP cocrystal, Form 1 , in the X-ray powder diffraction pattern have the values shown in bold (±0.05A).Example 15

[0110] Lutein-DL-a-Tocopherol cocrystal, Form 1 is characterized by XRPD peaks at about 12.11 , 18.18, 19.08, and 21.27 °20 ± 0.2 °20. It may be further characterized by XRPD peaks at about 15.57, 16.23, 18.58, 20.84 °20 ± 0.2 °20. Lutein-DL-a-tocopherol cocrystal, Form 1 , may also be characterized by a DSC endotherm at about 165.43°C. The lutein and the DL-a-tocopherol are preferably in a 1 :1 stoichiometric ratio. The cocrystal may also be characterized by an XRPD pattern and DSC thermogram such as depicted in Figures 16 and 17, respectively. Table 11 provides the d-spacing in A and the corresponding 20 values of lutein-DL-a-tocopherol cocrystal, Form 1.

[0111] The DL racemic mixture was used in these experiments because it is readily available, but other mixtures of tocopherol diastereomers or even pure stereoisomers (e.g., RRR-a-tocopherol) may also be used; it would be expected that the cocrystal characteristics described here for a racemic mixture may vary slightly with other mixtures or with isolated enantiomers.Table 11 : X-ray powder reflections (up to 33° 20) and intensities (normalized) of lutein- DL-a-tocopherol cocrystal, Form 1 . The value 20 [°] represents the diffraction angle in degrees and the value d [A] represents the specified distances in A between the latticeplanes. Particularly characteristic peaks for lutein- DL-a-tocopherol cocrystal, Form 1, in the X-ray powder diffraction pattern have the values shown in bold (±0.05A).Example 16. A Method for producing Lutein co-crystals

[0112] Following is a description of one method for producing lutein cocrystals. Other methods known in the art for forming cocrystals may also be employed, using lutein and one or more of the same coformers: a) mixing lutein with a cocrystal-forming compound (coformer) selected from the group consisting of gallic acid, gentisic acid, 5-HTP, and DL-a- tocopherol, typically (but not necessarily) in a 1 :1 molar ratio; b) moistening the mixture from step a) with an organic solvent (e.g., methanol or ethanol); c) subjecting the moistened mixture from step b) to grinding, such as grinding with a ball-mill (or other grinding apparatus known in the art); d) drying and collecting the cocrystals from step c).Example 17. Examples of Uses for the Polymorph or Co-crystal forms of Lutein

[0113] The present invention is also directed to a crystal or co-crystalline form of lutein as described herein for medical use.

[0114] The present invention is also directed to the use of a crystal or co-crystalline form as described herein for the treatment or prevention the ocular conditions described herein (e.g., cataracts, macular degeneration, photooxidative damage to retinal pigment epithelium, etc.)

[0115] The present invention is also directed to method for treating or preventing such a condition, said method comprising administering a therapeutically effective amount of a crystal or co-crystalline form as described herein to a human or other mammalian patient in need of such treatment or prevention.

[0116] The present invention is also directed to the use of a crystal or co-crystalline form as described herein for the manufacture of a medicament for the treatment or prevention of such a condition.Example 18: Grinding method for preparing lutein-gallic acid cocrystal, Form 1

[0117] 10 mg scale, 1:1 molar ratio. Lutein (10 mg, 0.018 mmol) was mixed with gallic acid (3.4 mg, 0.019 mmol) and 40 pL ethanol. The lutein : gallic acid molar ratio was ~1 :1. Grinding was carried out at room temperature for 60 min at 30 Hz, using aRetsch ball mill. After grinding, the materials were air-dried, harvested, and analyzed by XRPD, which indicated the formation of lutein-gallic acid cocrystals, Form 1.

[0118] 50 mg scale, 1:1 molar ratio. Lutein (50 mg, 0.088 mmol) was mixed with gallic acid (17 mg, 0.099 mmol) and 100 pL ethanol. The lutein : gallic acid molar ratio was slightly less than 1 :1. Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were air-dried, harvested, and analyzed by XRPD, which indicated the formation of lutein-gallic acid cocrystals, Form 1.Example 19: Grinding preparation for Lutein-Gentisic acid cocrystal, Form 1

[0119] 10 mg scale, 1:1 molar ratio. Lutein (10 mg, 0.018 mmol) was mixed with gentisic acid (3.0 mg, 0.019 mmol) and 40 pL ethanol. The lutein : gentisic acid molar ratio was ~1 :1 . Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the materials were air-dried, harvested, and analyzed by XRPD, which indicated the formation of lutein-gentisic acid cocrystals, Form 1 .

[0120] 50 mg scale, 1:1 molar ratio. Lutein (50 mg, 0.088 mmol) was mixed with gallic acid (14.9 mg, 0.096 mmol) and 100 pL ethanol. The lutein : gentisic acid molar ratio was slightly less than 1 :1. Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were airdried, harvested, and analyzed by XRPD, which indicated the formation of lutein- gentisic acid cocrystals, Form 1.Example 20: Grinding method for preparing Lutein-5-HTP cocrystal, Form 1

[0121] 10 mg scale, 1:1 molar ratio. Lutein (10 mg, 0.018 mmol) was mixed with5-hydroxytryptophan (4.4 mg, 0.019 mmol) and 40 pL ethanol. The lutein : 5- hydroxytryptophan molar ratio was ~1 :1 . Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were air-dried, harvested, and analyzed by XRPD, which indicated the formation of lutein-5-HTP cocrystals, Form 1 .

[0122] 50 mg scale, 1:1 molar ratio. Lutein (50 mg, 0.088 mmol) was mixed with5-hydroxytryptophan (21.9 mg, 0.099 mmol) and 100 pL ethanol. The lutein: 5- hydroxytryptophan molar ratio was slightly less than 1 :1. Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were air-dried, harvested, and analyzed by XRPD, which indicated the formation of Lutein-5-HTP cocrystals, Form 1 .Example 21 : Grinding method for preparing lutein-DL-g-tocopherol cocrystal, Form 1

[0123] 10 mg scale, 1:1 molar ratio. Lutein (10 mg, 0.018 mmol) was mixed withDL-a-tocopherol (9.14 pL, 0.020 mmol) and 40 pL ethanol. The lutein : DL-a- tocopherol molar ratio was ~1 :1 . Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were airdried, harvested, and analyzed by XRPD, which indicated the formation of lutein-DL- a-tocopherol cocrystals, Form 1 .

[0124] 50 mg scale, 1:1 molar ratio. Lutein (50 mg, 0.088 mmol) was mixed withDL-a-tocopherol (45.7 pL, 0.10 mmol) and 100 pL ethanol. The lutein : DL-a- tocopherol molar ratio was slightly less than 1 :1. Grinding was carried out at room temperature for 60 min at 30 Hz, using a Retsch ball mill. After grinding, the resulting materials were air-dried, harvested, and analyzed by XRPD, which indicated the formation of lutein-DL-a-tocopherol cocrystals, Form 1 .Example 22: In vitro diffusion test for lutein cocrystals embedded in bioadhesive hydrogel

[0125] In vitro lutein permeability tests were carried out with the novel co-crystalline forms of lutein using a bioadhesive hydrogel, as otherwise described in Bodoki E., Vostinaru O., Samoila O., Dinte E., Bodoki A.E., Swetledge S., Astete C.E., Sabliov C.M., Topical nanodelivery system of lutein for the prevention of selenite-induced cataract, Nanomedicine. 2019 Jan;15(1 ):188-197. Experiments were conducted in duplicate, without exposure to natural light, using a Franz cell diffusion system (PermeGear, Bechenheim, Germany), and synthetic hydrophilic polyethersulfone (PES) membranes (pore size 0.45 pm, Sigma). The Franz cells had a diffusion surface of 0.6362 cm2and 10 mL of receiving solution. 50mM TRIS buffer (pH = 7.1 ) with 1% (w / v) SDS was used as a receiving medium to confer sink conditions. The system was maintained at 37°C throughout the experiments with a thermostatic circulation bath (Julabo Corio C-B, Julabo GmbH, Seelbach, Germany). The receiving solution was stirred continuously (500 rpm) with a magnetic stirrer. The synthetic membrane was positioned between the donor and receiver compartments of the diffusion cells, whichhad previously been maintained for 30 min in the receiving solution to equilibrate. The volume of one drop of hydrogel was approx. 0.012 mL, with a density of 1.027 g / mL. Then 100 pL aliquots of the receiving solution were taken at various times (up to 48 h), always replenishing with fresh receiving medium. The lutein concentration in the collected samples was assessed by reverse-phase HPLC-UV (see Figure 9).

[0126] Each of the novel crystalline and co-crystal forms of lutein tested had a significantly improved diffusion profile as compared with that of unprocessed lutein (113 ng / mL within 48 h). The measured levels of lutein in the receiving media after 48 hours were 2368 ng / mL for lutein-gallic acid CC, 1587 ng / mL for lutein-gentisic acid CC, 1863 ng / mL for lutein-5-HTP CC, and 1587 ng / mL for lutein-D,L-tocopherol CC. A nearly 7-fold higher lutein concentration was observed in the receiving compartment for crystalline lutein (783 ng / mL within 48 h) as compared with the unprocessed lutein. Significantly higher levels of lutein were observed for all novel lutein co-crystals tested, with up to 20-fold higher levels for the lutein-gallic acid CC (2368 ng / mL within 48 h) as compared with the unprocessed lutein. Such enhancements in the effective solubility of lutein in aqueous media will improve the bioavailability of lutein, including its bioavailability following oral or topical administration.Methods of analysisX-ray powder diffraction (XRPD)

[0127] XRPD measurements were performed with a Bruker D8 Advance X-ray powder diffractometer at room temperature, using a copper filled X-ray tube (40 kV x 40 mA) as X-ray source, CuKa (A = 1.5418 A), a 0.6 mm divergence slit, 2.5° Soller slits on both the primary and secondary beams, and a 1 -dimensional LynxEye detector (with aperture angle of 2.91593°). Data were collected in the range 3.8-33° 20 with 0.02° increment steps, at a scan speed of 0.3 s / step.Differential Scanning Calorimetry (DSC)

[0128] Differential scanning calorimetry (DSC) was performed with a Shimadzu DSC-60 calorimeter in the range 20-300°C, with a heating rate of 10°C min-1using a crimped aluminum sample cell, and nitrogen flow of 60 mL / min.High-performance liquid chromatography (HPLC) with UV detection

[0129] Liquid chromatographic assays were conducted with an Agilent series 1200 LC system equipped with a DAD detector. Lutein was separated on a C8- based reverse phase column (4.6 x 50 mm, 5 pm) at 40°C, using a mixture of 80% ACN and 20% formic acid 0.1 % as the mobile phase. The injection volume was 50 pL and the detection wavelength was 446 nm. The flow rate was 0.8 mL / min (isocratic).Observations on the Cocrystallization of LuteinExample 23: Co-former selection

[0130] Various potential co-formers with lutein were screened to seek new cocrystal forms, for example for in vitro and in vivo testing on animal models of degenerative eye diseases. When designing crystals to have particular, desired properties, it is helpful to have a detailed understanding of the various molecular descriptors and their impacts, especially on intermolecular interactions that affect crystal structures. The general shape of a molecule is largely determined by its molecular conformation. Thus different conformers of the same molecule can produce different crystal packings. The conformational analysis employed molecular modeling of the lutein molecule, particularly the effects of the hydroxyl groups, followed by analysis of molecular similarity and selection of potential coformers based on their respective tendencies to form hydrogen bonds. Selections of potential coformers took account of functional groups with the potential to form supramolecular synthons. Finally, validation was carried out by comparison with conformations of other, known crystal structures. Several potential co-former candidates were thus identified, which we grouped into five functional classes: carbonyls (namely amides and ketones), carboxylic acids, amino acids, primary or secondary amines, and oligosaccharides. An additional factor that we considered was the potential synergistic effect of a candidate molecule with lutein, to boost antioxidant efficacy, or to help protect lutein against oxidative damage. Other desirable factors included ocular tolerance and low toxicity. In all cases a 1 : 1 stoichiometric ratio of Lutein : candidate co-former was used. We explored multiple crystallization methods, including cooling-evaporative crystallization,grinding assisted by solvent, and slurry preparations. These experiments identified, from a much larger group of initial candidates, 15 potential coformers that were identified for further investigation. Of these, four were considered particularly promising, namely gallic acid, gentisic acid, 5-HTP, and DL-alpha- tocopherol. The results, shown in Table 12, underscore the importance of careful selection of potential coformers.Table 12. HPLC data and the calculated amount of Lutein in the 15 candidate co-crystals.Co-former Molar ratio Peak Lutein %(CCF) SM / C.LArea RT Corr Area lJl'molar ratio 3990.6 8.081 493.83 58.87Gentisic acid 1:1 molar ratio 7613.6 8.074 942.98 94.81PPVA 1:1 weight ratio 3677.5 8.09 454.57 71.92Gallic acid 1:1 molar ratio 7265.7 8.076 899.67 92.45PLGA 1:1 weight ratio 3234.5 8.017 403.46 63.83Betaine 1:1 molar ratio 5349.9 8.013 667.65 63.69Piperazine 1:1 molar ratio 7136.5 8.011 890.84 81.14Pyrazine 1:1 molar ratio 7095.4 8.015 885.27 79.895-HTP 1:1 molar ratio 6443 8.018 803.57 88.18DHA 1:1 molar ratio 2541.3 7.974 318.70 39.77DL-a-Tocopherol 1:1 molar ratio 5933.2 8.014 740.35 102.91Vascepa 1:1 molar ratio 1441.3 8.055 178.93 22.38 lJl'molar ratio 249038 8.058 309.1 1 37.45L-Proline 1:1 molar ratio 3421.6 9.34 366.34 34.85Trehalose 1:1 molar ratio 2846.4 9.355 304.27 38.55Example 24: Electron diffraction-based crystal structure determination

[0131] The structure of crystalline lutein was determined at room temperature with an ELDICO ED-1 electron diffractometer equipped with a LaB6 source operating at 160 kV (A = 0.02851 A), producing a parallel beam with a diameter of 750 nm, and using a QUADRO hybrid-pixel detector from DECTRIS. A small amount of powder was gently ground between two glass slides. A carbon-coated 300-mesh Cu grid was placed on top of the sample and gently pressed again with a clean glass slide. Using an optical microscope, it was verified that the groundsolid had been properly deposited onto the sample grid. The sample grid was mounted on a motorized stage that allowed for translation and rotation with submicrometer precision. An electron beam, operated in scanning transmission electron microscopy (STEM) mode, enabled the inspection and imaging of the sample to identify suitable nanocrystalline particles. A sub-micron crystal was thus chosen and aligned in the center of the beam. Diffraction data were then collected in 0.5° increments per frame as the crystal continuously rotated at 1 ° per second over a 120° range. The data were analyzed by CrysAlisPro software for determination of the unit cell and to extract the integrated intensities. The crystal structure was solved with Olex2 software.Example 25: Crystal structure elucidation through electron diffraction (ED)

[0132] The preparation of single lutein crystals is difficult due to lutein’s complex structure, which includes both a conjugated polyene chain, and bulky hydroxyl groups that hinder regular molecular packing. Lutein's hydrophobic nature and its low water solubility complicate the search for suitable solvents for growing crystals. Also, lutein is sensitive to the presence of impurities and oxidation, which further impede crystallization. Nucleation tends to be slow. Crystal growth is challenging. Prior approaches to crystallizing lutein have often produced only small, poorly- formed crystals. Standard crystallization techniques can be less effective for lutein, and thus advanced techniques can be employed such as vapor diffusion, microseeding, or the use of crystallization promoters.

[0133] The newly-discovered crystalline forms of lutein appear to show no similarity with other crystalline structures of lutein that had been previously reported. See Figure 18. For example, comparing the conformation of the conjugated polyene chain in the new crystalline form with that from lutein solvates revealed that the crystalline Lutein has a more fixed conformation, whereas in the solvated structures with methanol or ethanol, interactions between lutein and the solvents lead to structural changes, particularly at the ends of the polyene chain. These solvent interactions evidently cause the lutein molecule to adopt different conformations, leading to potential twisting or bending of the solvated molecule, in contrast to the more fixed crystalline form.

[0134] The structure of the novel crystalline lutein was determined by electron diffraction (ED), using a single diffraction dataset from a single nanocrystal. We found that the lutein had a triclinic crystallographic system, space group P-1 , with a unit cell volume of V = 929.5 A3. Notably, the asymmetric unit contains 0.5 molecules of Lutein (Z’ = 0.5), while the entire unit cell accommodates one molecule of crystalline Lutein (Z = 1 ). Table 13 presents crystallographic data for the crystalline lutein from ED. The similarity between the calculated XRPD pattern, based on the determined crystal structure, and what was experimentally observed supports the validity of the ED-derived structure for lutein, a structure that is representative of the bulk material. See Fig. 19.Table 13. Crystallographic data for the novel crystalline lutein, as determined by ED. Single-crystal data (low temperature) of the solvates, as reported by Guo et al.(2021), are also shown for comparison.Lutein Ethanol Lutein MethanolCrystalline Lutein solvate solvate(AKEQAL) (AKEGEF)Method ED SC-XRI) SC-XRDStructural formula C40H56O2 C44H68O4 C42H61O4Formula weight 568.84 660.98 629.9(g / mol)Temp (K) 293 (2) 113 113Crystal system Triclinic Triclinic TriclinicSpace group P (-1) P (-1) P (-1)Cell length a (A) 8.363(4) 8.4891(17) 8.2931(68) b (A) 9.019(3) 10.273(2) 9.0677(73) c (A) 12.943(5) 13.108(3) 13.6180(110) a (°) 81.05(4) 94.10(3) 91.2098(151) p (°) 74.61(4) 108.09(3) 91.2916(118) y (°) 86.32(3) 101.65(3) 94.2400(151)Cell volume (A3) 929.5(7) 1053.1(4) 1020.7(4)1, 1' Z: 1, Z': 0.5 Z: 1, Z': 0.5 Z: 1, Z': 0.5Calculated density1.016 1.009 0.9908(g / cm3)

[0135] The observed crystal packing of the new crystalline Lutein form featured only very small solvent-accessible voids, suggesting that the lutein molecules pack together efficiently in the crystal lattice, leaving only minimal unoccupied spacesand maximizing molecular interactions, thereby contributing to the stability and rigidity of the crystalline structure. The accessible voids in the crystalline lutein structure (with a probing radius of 3.60 A3) accounted for only 0.4% of total unit cell volume. A volume of approximately 30 A3is required to accommodate a single water molecule, and thus the accessible space appears to be insufficient to entrap any water or other solvent molecules in the crystal structure.

[0136] By contrast, the ethanol and methanol solvates showed no accessible solvent voids within their crystal packing. However, when the solvent molecules were omitted, the remaining crystal structure exhibited a solvent-accessible surface volume of 23.72 A3for the methanol solvate, and 39.90 A3for the lutein ethanol solvate. These larger voids are due to the accommodation of the respective solvent molecules within the crystal lattices. The novel structure is thus clearly distinct from these prior solvate structures.Discussion

[0137] Several biochemical mechanisms are believed to be involved in the formation of cataracts, including loss of calcium homeostasis, calpain-induced proteolysis, crystallin precipitation, and cytoskeletal loss. Lutein’s antioxidant properties could help to inhibit at least some of these pathways. The novel crystalline and co-crystalline formulations of lutein, especially when complemented with a bioadhesive formulation, enhance the ocular bioavailability of lutein and increase its therapeutic efficacy.

[0138] Future experiments with animal models of macular degeneration will confirm the efficacy of the novel lutein compositions for preventing or inhibiting the progress of macular degeneration. Models of age-related macular degeneration (AMD) have been developed, for example, in mice, rats, rabbits, pigs, and non-human primates. See for example Penessi ME, Neuringer M, Courtney RJ. Animal models of age-related macular degeneration. Mol Aspects Med. 2012, 33(4): 487-509. There are at least four rodent models of macular degeneration. One model relies on an inactivated SOD1 gene (SOD1- / - mice). See Imamura Y, Noda S, Hashizume K, Shinoda K, Yamaguchi M, Uchiyama S, Shimizu T, Mizushima Y, Shirasawa T, Tsubota K. Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1 -deficient mice: a model of age-related macular degeneration. Proc. Nat. Acad. Sci. USA. 2006; 103(30): 11282-11287. Another model relies on an inactivated ApoE gene (ApoE - / -mice). See Dithmar S, Sharara NA, Curcio CA, Le NA, Zhang Y, Brown S, Grossniklaus HE. Murine high-fat diet and laser photochemical model of basal deposits in Bruch membrane. Arch. Ophthalmol. 2001 ; 119(11 ): 1643— 1649. A different type of model relies on aging mice (16 months) fed a high-fat diet. See Cousins SW, Espinosa-Heidmann DG, Alexandridou A, Sall J, Dubovy S, Csaky K. The role of aging, high fat diet and blue light exposure in an experimental mouse model for basal laminar deposit formation. Exp. Eye Res. 2002; 75(5):543-553. Still another model relies on ultraviolet induction of macular degeneration. See Pavelic SC et al. UV-induced retinal proteome changes in the rat model of age-related macular degeneration. Biochimica et Biophysica Acta-Molecular Basis of Disease. 2015, 1852 (9):1833-1845. The first two models may be better suited for testing the effect of lutein on potential disease remission. The last two models may be better suited for testing effect of lutein in protecting against the development of AMD. In each case, the lutein formulation would preferably be administered in a bioadhesive hydrogel, topically applied to the cornea.

[0139] In one embodiment, an in situ bioadhesive gel for use with the lutein formulations comprises a mixture of 2.7% (w / w) bioadhesive polymer (polyethylene oxide, Polyox™ 1105, Dow Chemical, MW -900,000), and 16.5% (w / w) Poloxamer P407 (a triblock copolymer comprising a central hydrophobic block of polypropylene glycol, flanked by two hydrophilic blocks of polyethylene glycol; the approximate lengths of the two PEG blocks is 101 repeat units, and the approximate length of the propylene glycol block is 56 repeat units). The polyethylene oxide - Poloxamer P407 mixture readily forms a thermoreversible gel. The Polyethylene oxide 1105 and the Poloxamer P407 are each separately dispersed in sterile water until used. The Polyethylene oxide / Poloxamer mixture is prepared by mixing the dispersions, and the mixture is stored in a refrigerator (4°C) until used. The lutein composition is later added to the bioadhesive in situ gel forming matrix under continuous, gentle stirring.

[0140] The bioadhesive matrix may optionally comprise another polymer, copolymer, or mixture of polymers or copolymers with bioadhesive properties, including for example polyacrylic acid derivatives, cellulose derivatives, polycarbophil, other polyethylene oxides, hyaluronic acid derivatives, pectin, carrageenan, alginates, and the like. It is preferred that the matrix should be bioadhesive, that it should be thermosensitive (to form a gel and release lutein slowly at body temperature, or more specifically the temperature of the conjunctival sac), that it should be well-tolerated by ocular mucosa, that it should be compatible with the lutein polymorph or cocrystal, thatit should facilitate controlled and reproducible release of the dispersed bioactive ingredient, and that it should exhibit prolonged retention following topical administration.

[0141] A preferred bioadhesive matrix combination of poloxamer and polyethylene oxide provides several beneficial properties: Poloxamer is compatible with the ocular mucosa. Poloxamer is a thermoreversible polymer which, at higher concentrations and temperatures, forms a stable, rigid gel that would by itself be difficult to apply topically. At lower temperatures, the polymer stays in aqueous solution - a liquid. As the temperature rises, the polymer forms a gel. Preferably the composition is a liquid at room temperature, but becomes a gel at body temperature (or more specifically, at the temperature of the conjunctival sac, which may be 2-3 degrees below body temperature), allowing for the slow release of the active ingredient once the composition forms a gel on the surface of the cornea. Polyethylene oxides have good adhesive qualities. Polyethylene oxide 1105 (with a small to medium molecular weight range) is a preferred compound due to its rheological characteristics. Polyethylene oxides are also compatible with ocular mucosa. A mixture of poloxamer and polyethylene oxide provides a product that is easily applied as a liquid to the cornea, into the conjunctival sac, and that forms a gel following contact with the conjunctiva at body temperature. The mixture has enhanced bioadhesive properties, for extended retention following topical administration, and thus improved bioavailability of lutein to the eye - including the interior of the eye and the retina. These nonionic polymers should be compatible with the bioactive components.

[0142] Although it is preferred to administer the lutein compositions as drops to form a thermoreversible gel as described, other routes of administration may also be used. Other pharmaceutical formulations otherwise known in the art may optionally be used - e.g., liquid eye preparations (eye drops, eye lotions, gel-forming solutions); or semisolid eye preparations (ointments, gels); solid eye preparations (powders, ocular inserts); or aerosols (ophthalmic drugs mixed with a gas under pressure); or ocular inserts such as contact lenses.

[0143] Through routine experimentation to test various proportions, the ratios and concentrations of the various components are optimized to enhance residence time on and penetration into the cornea. Typical expected ranges include polyethylene oxide 1105 in a range of 1 .5-3.5% (w / w), and Poloxamer P407 in a range of 12-19% (w / w).

[0144] The bioadhesive matrix should produce a good dispersion of the lutein composition, it should have sufficient viscosity to maintain homogeneity during storage (physical stability), it should allow ready application on conjunctival mucosa, and it should be compatible with the particular lutein composition being used.

[0145] Depending on the concentration of lutein in the ophthalmic preparation, the bioadhesive / thermosensitive polymer’s concentration can be adjusted to optimize viscosity and bioadhesive capacity. Expected lutein loadings in the bioadhesive gel include the range 1-5% w / v%. Higher and lower concentrations can also be used.

[0146] Other bioadhesive gel-forming matrices may be prepared by mixing suitable polymers in appropriate proportions. Examples of bioadhesive polymers include one or more of polyacrylic acid, polycarbophil, polyethylene oxides, cellulose derivatives, hyaluronic acid derivatives, pectin, carrageenan, alginates, and the like. The molecular weight may be chosen to optimize performance.

[0147] In future embodiments, an alternative means of administration will be to incorporate a lutein-containing composition as described in any aspect of this disclosure into an ocular insert or contact lens, wherein the ocular insert or contact lens then releases lutein to a mammalian eye over time.Miscellaneous

[0148] As used in the specification and claims, a “therapeutically effective amount” of a composition refers to a quantity of the composition sufficient to be therapeutically effective to prevent, inhibit, slow the progression, or treat the symptoms of a disease, for example cataracts, dry macular degeneration or wet macular degeneration (age- related macular degeneration), Stargardt disease, or retinitis pigmentosa. Where appropriate in context, a “therapeutically effective amount” of a composition can also refer to a quantity of the composition that, when administered topically to a tissue, is sufficient to deliver a concentration of lutein to the tissue to have a clinically meaningful effect on the tissue or neighboring tissues.

[0149] The complete disclosures of all references cited in this application are hereby incorporated by reference. Also incorporated by reference is the complete disclosure of the priority application, United States provisional patent application serial number 63 / 598,201 , filed 13 November 2023. In the event of an irreconcilable conflict, the present disclosure takes priority over the disclosure of material incorporated by reference.

Claims

What is claimed:

1. A composition of matter comprising lutein; wherein said composition comprises one or more lutein crystals, or one or more lutein co-crystals, or both; wherein said lutein crystals, if present, have an x-ray powder diffraction pattern comprising peaks at 10.87, 11.28, 12.94, 14.12, 14.48, 14.90, 15.58, 16.12, 17.11 , 18.51 , 21.49, 22.74, 24.19, and 24.32 °20 ± 0.2 °20; and wherein said lutein co-crystals, if present, comprise lutein and a co-crystal- forming compound selected from the group consisting of gallic acid, gentisic acid, 5-hydroxytryptophan, and a tocopherol.

2. The composition of Claim 1 , wherein said composition comprises one or more cocrystals comprising: a) lutein; and b) a co-crystal-forming compound selected from the group consisting of gallic acid, gentisic acid, 5-hydroxytryptophan, and a tocopherol.

3. The composition of Claim 2, wherein the cocrystal-forming compound is gallic acid.

4. The composition of Claim 3, wherein said cocrystal has an x-ray powder diffraction pattern comprising peaks at 6.46, 17.25 and 19.15 °20 ± 0.2 °20.

5. The composition of Claim 4, wherein said cocrystal has an x-ray powder diffraction pattern additionally comprising one or more peaks selected from the group consisting of 7.20, 13.64, 14.31 , 15.63, 16.19, 20.24, 21.49, 24.36, and 25.95 °20 ± 0.2 °20; and a melting point of 154°C.

6. The composition of Claim 2, wherein the cocrystal-forming compound is gentisic acid.

7. The composition of Claim 6, wherein said cocrystal has an x-ray powder diffraction pattern comprising peaks at 10.95, 15.71 , 16.15, 17.51 , and 24.80 °20 ± 0.2 °20.

8. The composition of Claim 7, wherein said cocrystal has an x-ray powder diffraction pattern additionally comprising one or more peaks selected from the group consisting of 14.34, 14.64, 15.01 , 17.25, 18.65, 21.60, and 24.40 °20 ± 0.2 °20; and a melting point of 140°C.

9. The composition of Claim 2, wherein the cocrystal-forming compound is 5- hydroxytryptophan.

10. The composition of Claim 9, wherein said cocrystal has an x-ray powder diffraction pattern comprising peaks at 11.08, 14.22, 16.41 , 17.49, 21.04, 23.62, 25.32, 25.70, and 28.20 °20 ± 0.2 °20.

11. The composition of Claim 10, wherein said cocrystal has an x-ray powder diffraction pattern additionally comprising one or more peaks selected from the group consisting of 14.59, 14.96, 15.66, 16.18, 17.26, 18.52, 19.17, 21 .56, 22.77, and 24.36 °20 ± 0.2 °20; and a melting point of 173°C.

12. The composition of Claim 2, wherein the cocrystal-forming compound is DL- a-Tocopherol.

13. The composition of Claim 12, wherein said cocrystal has an x-ray powder diffraction pattern comprising peaks at 12.11 , 18.18, 19.08, and 21.27 °20 ± 0.2 °20.

14. The composition of Claim 13, 15.57, 16.23, 18.58, and 20.84 °20 ± 0.2 °20; and a melting point of 165°C.

15. A process for making the composition of Claim 2, said process comprising the steps of: a) mixing lutein with a cocrystal-forming compound selected from the group consisting of gallic acid, gentisic acid, 5-hydroxytryptophan, and a tocopherol, in a molar ratio of lutein to the cocrystal-forming compound of about 1 :1 ; b) moistening the mixture from step (a) with an organic solvent; c) grinding the moistened mixture from step (b); and d) evaporating the solvent from the ground mixture from step (c).

16. The composition produced by the method of claim 15.

17. The composition of Claim 1 , wherein said composition comprises crystalline lutein having an x-ray powder diffraction pattern comprising peaks at 10.87, 11.28, 12.94, 14.12, 14.48, 14.90, 15.58, 16.12, 17.11 , 18.51 , 21.49, 22.74, 24.19, and 24.32 °20 ± 0.2 °20.

18. The composition of Claim 17, wherein said crystalline lutein has an x-ray powder diffraction pattern additionally comprising one or more peaks selected from the group consisting of 7.05, 9.78, 17.95, 19.79, 20.02, 26.09, 26.57, 27.16 °20 ± 0.2 °20; and a melting point of 175.8°C.

19. The composition of Claim 17, wherein said crystalline lutein has the following crystallographic properties, as measured by powder x-ray diffraction at a temperature of about 294°K: a P-1 space group, unit cell dimensions of a=8.363(4) A, b=9.019(3) A, c=12.943(5) A, a =81.05(4) °, 0=74.61(4) °, y =86.32(3)°.

20. A process for making the composition of Claim 17, said process comprising the steps of: a) mixing lutein with a solvent comprising one or more compounds selected from the group consisting of methanol, ethanol, another alcohol, and a mixture of an alcohol and water; b) heating the mixture from step a) to a temperature of about 40°C for at least about 30 minutes; c) allowing the mixture from step b) to settle at a temperature of about 5°C; d) separating the solid phase from step c), and e) evaporating the solvent from the separated solid phase from step d).

21. Crystalline lutein produced by the method of Claim 20.

22. A process for making the composition of Claim 17, said process comprising the steps of: a) moistening solid lutein with a solvent comprising one or more compounds selected from the group consisting of methanol, ethanol, another alcohol, and a mixture of an alcohol and water; b) grinding the moistened lutein; and c) evaporating the solvent from the ground, moistened lutein to produce a crystalline lutein.

23. Crystalline lutein produced by the method of Claim 22.

24. A method for treating or preventing a condition selected from the group consisting of cataracts, diabetic retinopathy, age-related macular degeneration, and photooxidative damage to retinal pigment epithelium, said method comprising administering to a mammalian patient a therapeutically effective amount of a composition as recited in Claim 1.

25. The method of Claim 24, wherein the composition comprises one or more cocrystals comprising lutein; and a co-crystal-forming compound selected from the group consisting of gallic acid, gentisic acid, 5-hydroxytryptophan, and a tocopherol.

26. The method of Claim 24, wherein the composition comprises crystalline lutein having an x-ray powder diffraction pattern comprising peaks at 10.87, 11.28, 12.94, 14.12, 14.48, 14.90, 15.58, 16.12, 17.11 , 18.51 , 21.49, 22.74, 24.19, and 24.32 °20 ± 0.2 °20.

27. A method for delivering lutein to the eye of a mammal, wherein the mammal has or is at risk for developing cataracts, or macular degeneration, or photooxidative damage to retinal pigment epithelium; said method comprising topically administering to the mammal’s eye a composition that comprises a mixture of a composition as defined in any of the above composition claims, and a hydrogel:(a) wherein the hydrogel comprises water, a thermoreversible gel-forming polymer, and a bioadhesive polymer; wherein the mixture is liquid at 25°C; wherein the thermoreversible gel-forming polymer causes the mixture to become a gel at the temperature of the conjunctival sac or of the surface of the cornea of the mammal; and wherein the bioadhesive polymer causes the mixture to adhere to the conjunctival mucosa and cornea more strongly than the mixture would adhere without the bioadhesive polymer;(b) wherein the mixture is applied as a liquid to the surface of the cornea or into the conjunctival sac; wherein the temperature of the conjunctival sac or of the surface of the cornea causes the mixture to form a gel; wherein the mixture adheres to the conjunctival mucosa, to the surface of the cornea, or both; and wherein the adhering gel releases lutein to the eye over a period of time; and(c) wherein the mammal has or is at risk for developing cataracts, or macular degeneration, or photooxidative damage to retinal pigment epithelium.

28. The method of Claim 24, wherein the thermoreversible gel-forming polymer comprises a poloxamer.

29. The method of Claim 24, wherein the bioadhesive polymer comprises a polyethylene oxide.

30. An ocular insert or contact lens comprising a composition as in any of the above composition claims, wherein the ocular insert or contact lens releases lutein to a mammalian eye over time.

Images