Semi-fluorinated compounds for ophthalmic administration
阅读说明：本技术 用于眼部给药的半氟化化合物 (Semi-fluorinated compounds for ophthalmic administration ) 是由 迪特尔·谢勒 拉尔夫·格里伦恩伯杰 弗兰克·洛施尔 哈特穆特·沃斯 于 2016-09-29 设计创作，主要内容包括：本发明涉及用于眼部给药的半氟化化合物,具体涉及包含以通式CF-(3)-(CF-(2))-(n)-CH(CH-(3))-(CH-(2))-(m)-CH-(3)表征的半氟化化合物的眼用组合物,其中n是选自3至5的整数,且m是选自1至5的整数。本发明还提供所述组合物作为药物局部施用于眼的用途,以及一种试剂盒,其包含所述组合物和容器,所述容器包括分配装置,例如滴管。(The present invention relates to semifluorinated compounds for ophthalmic administration, in particular to compositions comprising a compound of formula CF 3 ‑(CF 2 ) n ‑CH(CH 3 )‑(CH 2 ) m ‑CH 3 An ophthalmic composition of a semifluorinated compound characterized, wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5. The invention also provides said composition as a medicamentFor topical application to the eye, and a kit comprising the composition and a container comprising a dispensing device, such as a dropper.)
1. An ophthalmic composition comprising a compound characterized by the general formula:
CF3-(CF2)n-CH(CH3)-(CH2)m-CH3
wherein n is an integer selected from 3 to 5, and m is an integer selected from 1 to 5.
2. The composition of claim 1, wherein n is 5 and m is 5, or wherein n is 3 and m is 2.
3. The composition according to any one of claims 1 or 2, which is in liquid form and comprises at least 1 wt% of the compound, in particular from 1 wt% to 5 wt% of the compound, based on the total weight of the composition.
4. The composition of any one of the preceding claims, formulated as a clear liquid solution.
5. The composition of any one of the preceding claims, which is substantially free of:
(a) a polymer,
(b) perfluorinated compounds, and/or
(c) A dissolved pharmacologically active ingredient that is not a semifluorinated alkane.
6. Composition according to any one of the preceding claims, having a dynamic viscosity at ambient temperature and pressure not exceeding 10 mPa-s, and preferably not exceeding 4 mPa-s.
7. The composition of any one of the preceding claims, consisting essentially of the compound and optionally one or more lipophilic liquid components.
8. A composition according to any one of the preceding claims for use as a medicament.
9. The composition of claim 8, wherein the composition is administered topically within the lacrimal sac, within the lower eyelid, on the surface of the eye, or on ocular tissue.
10. The composition according to claim 8 or 9 for use in the treatment of dry eye disease (keratoconjunctivitis sicca) and/or meibomian gland dysfunction or symptoms or conditions associated therewith.
Technical Field
The present invention is in the field of semi-fluorinated compounds, compositions thereof, and their use as pharmaceuticals, particularly for ophthalmic administration.
Background
Semifluorinated alkanes are compounds comprising at least one non-fluorinated hydrocarbon segment and at least one perfluorinated hydrocarbon segment. Described of the general formula CF3(CF2)n(CH2)mLinear, unbranched, semifluorinated alkanes of CH, where n and m are integers representing the number of carbon atoms of the respective segment, for various applications, such as commercially for unfolding and recoating the retina, long-term tamponades as vitreous fluid substitutes (h.meiert et al, European Journal of opthalmology, vol.10(3), pp.189-197,2000), and as eluates of residual silicone oils after vitreoretinal surgery.
Formula CF3(CF2)n(CH2)mCH3The semifluorinated alkanes of (a) are described in other applications.
WO2011/073134 discloses at formula CF3(CF2)n(CH2)mCH3Optionally in the presence of a co-solvent such as ethanol, wherein the semifluorinated alkane serves as a liquid drug delivery vehicle for the cyclosporin for the topical treatment of keratoconjunctivitis sicca.
WO2014/041055 describes the formula CF3(CF2)n(CH2)mCH3(which may optionally be denoted as F (CF)2)n(CH2)mH) Mixtures of semifluorinated alkanes of (a). These mixtures are described as being ophthalmically useful as tear film substitutes or for treating patients with dry eye and/or meibomian gland dysfunction.
Common use toolThe semifluorinated compound with linear and unbranched segments is named FnHm, where F represents the perfluorinated hydrocarbon segment, H represents the non-fluorinated segment, and n and m define the number of carbon atoms of the corresponding segment. For example, F3H3 is used for perfluoropropylpropane CF3(CF2)2(CH2)2CH3I.e., 1-perfluoropropylpropane.
However, of the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3The semifluorinated alkanes of (a) have not been described, in particular for ophthalmic applications. It is therefore an object of the present invention to provide such compounds, in particular in terms of their use in compositions, in particular in the field of ophthalmic applications.
Disclosure of Invention
In a first aspect, the present invention relates to a composition comprising a compound represented by the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3A composition of characterized semifluorinated compounds, wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5. Preferably, these compositions are in liquid form, wherein the composition comprises at least 1% by weight, in particular from 1% to 5% by weight, of the at least one compound selected from the group consisting of CF, beta-cyclodextrin, and mixtures thereof3-(CF2)n-CH(CH3)-(CH2)m-CH3A compound of characterization, wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5.
In another aspect, the invention relates to a composition comprising said compound in the form of a transparent liquid solution and also to a composition consisting essentially of said compound and optionally one or more lipophilic liquid components.
In yet another aspect, the invention provides the use of a composition comprising the compound for the treatment of dry eye and/or meibomian gland dysfunction and any symptoms or conditions associated therewith.
In another aspect, the present invention provides a method of treating dry eye and any symptoms or conditions associated therewith, comprising topically applying the composition to the lacrimal sac, into the lower eyelid, to the surface of the eye, or to ocular tissue.
In yet another aspect, the invention provides a kit comprising a composition of the invention in a container comprising a dispensing device suitable for topical administration of the composition to the eye or eye tissue.
Drawings
FIG. 1 is a drawing depicting a reaction from a compound CF3(CF2)5(CH2)7CH3And CF3-(CF2)5-CH(CH3)-(CH2)5-CH3Relative evaporation time of the constituent compositions and Compound CF3-(CF2)5-CH(CH3)-(CH2)5-CH3Percentage in the composition (indicated by the filled circles and dotted lines in the figure), and by the compound CF3(CF2)3(CH2)4CH3And CF3-(CF2)3-CH(CH3)-(CH2)2-CH3Relative evaporation time of the constituent compositions and Compound CF3-(CF2)3-CH(CH3)-(CH2)2-CH3Graph of percentage as a function of the composition (indicated by open circles and dashed lines in the graph).
FIG. 2 is a drawing depicting a reaction from a compound CF3(CF2)5(CH2)7CH3And CF3-(CF2)5-CH(CH3)-(CH2)5-CH3Measured refractive index of the constituent composition and Compound CF3-(CF2)5-CH(CH3)-(CH2)5-CH3A graph of the percentage as a function of the composition.
FIG. 3 is a drawing depicting a reaction from a compound CF3(CF2)3(CH2)4CH3And CF3-(CF2)3-CH(CH3)-(CH2)2-CH3Measured refractive index of the constituent composition and Compound CF3-(CF2)3-CH(CH3)-(CH2)2-CH3A graph of the percentage as a function of the composition.
FIG. 4 is a schematic representation of a mask containing CF3(CF2)5(CH2)7CH3And CF3-(CF2)5-CH(CH3)-(CH2)5-CH3The composition of (a), hyaluronic acid standard reference and 0.01% BAC positive control in vitro eye irritation test (EVEIT) compared the corneal erosion size measurement (mm) over the three days2) Schematic representation of (a).
Detailed Description
In a first aspect, the present invention relates to a catalyst of the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3A semifluorinated compound of the characterization, wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5.
Particularly preferred formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3Are those wherein n is 5 and m is 5 (i.e., formula CF)3-(CF2)5-CH(CH3)-(CH2)5-CH3) Or those in which n is 3 or m is 2 (i.e., formula CF)3-(CF2)3-CH(CH3)-(CH2)2-CH3). Other preferred compounds include CF3-(CF2)3-CH(CH3)-(CH2)3-CH3,CF3-(CF2)3-CH(CH3)-(CH2)5-CH3,CF3-(CF2)5-CH(CH3)-(CH2)1-CH3And CF3-(CF2)5-CH(CH3)-(CH2)3-CH3。
Alternatively, the compounds of the invention may also be generally referred to as 2-perfluoroalkylalkanes, e.g. compounds CF based on hydrocarbon alkanes as roots (roots)3-(CF2)5-CH(CH3)-(CH2)5-CH3It may also be referred to as 2-perfluorohexyloctane.
These compounds have a stereogenic center at the 2-alkyl position. As understood herein, the general formula includes both enantiomers, enriched mixtures of the two enantiomers, and racemic mixtures.
It has been found that the compounds as defined above provide a number of unexpected advantages, as described below. When provided as compositions, they are particularly useful, for example, for medical applications, particularly for topical administration, such as topical ocular administration.
The invention relates to a composition comprising a compound of formula CF as defined above3-(CF2)n-CH(CH3)-(CH2)m-CH3The compositions of these semifluorinated compounds characterized are preferably in liquid form, e.g., formulated for administration as a liquid solution. In optional embodiments, the composition may be formulated for administration as a gel, suspension, microemulsion, or spray. Preferably, the composition is provided in sterile form.
In a particularly preferred embodiment, comprises a compound represented by the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3(wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5) is in liquid form and comprises at least 1% by weight of said compound, in particular 1% to 5% by weight of said compound, based on the total weight of the composition. In other embodiments, the composition may comprise from about 3 wt% to 5 wt%, or from about 5 wt% to 10 wt%, or up to 25 wt% of the compound, based on the total weight of the composition.
In a preferred embodiment, the composition as defined above is formulated as a clear liquid solution. Transparent in this case means that there are no dispersed solid or liquid particles that cause turbidity. In other words, the clear solution is a pure single phase liquid system, with only small and technically irrelevant amounts of particulate impurities that may be present.
Furthermore, the composition is preferably formulated as a liquid solution that exhibits a refractive index close to that of water of 1.333 at Room Temperature (RT). In a particularly preferred embodiment, the refractive index of the liquid solution at 20 ℃ is in the range of about 1.30 to about 1.35 as measured by a refractometer.
The compositions as defined above may also contain other excipients as required or available, for example one or more acids, bases, electrolytes, buffers, solutes, antioxidants, stabilizers and, if desired, preservatives. In a preferred embodiment, the composition as defined above is substantially free of water and/or substantially free of preservatives, such as benzalkonium chloride (benzalkonium chloride).
In another preferred embodiment, the composition of the present invention is formulated as a clear liquid solution substantially free of: (a) a polymer, (b) a perfluorinated compound, and/or (c) a dissolved pharmacologically active ingredient that is not a semifluorinated alkane. In another embodiment, the composition as described herein may be substantially free of any form of pharmacologically active ingredient and which is not a semifluorinated alkane.
As understood herein, the term "substantially free" with respect to a component of a composition means that the component is present in an amount not exceeding trace amounts, and if present in trace amounts, the component does not contribute technically to the composition.
Examples of polymers that are preferably not present in the compositions of the present invention include silicone polymers (polymerized siloxanes), polyether polymers and fluorinated or perfluorinated derivatives thereof.
Examples of perfluorinated compounds (i.e., compounds in which all hydrogen atoms are substituted with fluorine and which are preferably absent in the compositions of the present invention) include perfluorinated alkanes, such as perfluorodecalin, and halogenated perfluorinated alkanes, such as perfluorooctylbromide.
The composition of the invention in the form of a transparent liquid solution is also substantially free of dissolved pharmacologically active ingredient (which is not a semifluorinated alkane). As used herein, the term "pharmacologically active ingredient" refers to any type of pharmaceutically active compound or drug, i.e., a pharmaceutically active compound or drug that produces a pharmacological effect and is therefore useful for prophylaxis, diagnosis, stabilization, treatment, or, in general, for management of a condition or disease.
However, the compounds of the present invention and compositions containing them have beneficial therapeutic effects at the site of administration even if they do not contain other pharmacologically active ingredients.
In a further preferred embodiment, the composition of the invention consists essentially of the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3(wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5) and optionally one or more lipophilic liquid components. In one embodiment, the composition consists essentially of the compound CF3-(CF2)5-CH(CH3)-(CH2)5-CH3Or CF3-(CF2)3-CH(CH3)-(CH2)2-CH3Or mixtures thereof, and optionally one or more lipophilic liquid components.
As used herein, the term "consisting essentially of is a so-called closed language meaning that only the components mentioned are present. Rather, the terms "comprising," "including," and "containing" are used herein as so-called open language, which means that other ingredients may also be present.
The optional lipophilic liquid component is preferably a substantially water-insoluble and/or water-immiscible excipient, for example an oily excipient such as a lipid, a triglyceride oil and any other physiologically tolerated oil of the eye or as e.g. of the general formula CF3(CF2)n(CH2)mCH3Wherein n and m are independently selected from integers of 3 to 8, e.g. CF3(CF2)3(CH2)4CH3Or CF3(CF2)5(CH2)7CH3。
Such lipophilic liquid ingredients may be present in the composition in an amount of up to about 25 wt.%, or up to about 50 wt.%, or 75 wt.%, or 90 wt.%, 95 wt.%, or 97 wt.% of the composition, based on the total weight of the composition.
In a further preferred embodiment, the composition according to the invention comprises CF3-(CF2)5-CH(CH3)-(CH2)5-CH3And CF3-(CF2)5-(CH2)7-CH3(ii) a Or comprises CF3-(CF2)3-CH(CH3)-(CH2)2-CH3And CF3-(CF2)3-(CH2)4-CH3Or consisting essentially of CF3-(CF2)5-CH(CH3)-(CH2)5-CH3And CF3-(CF2)5-(CH2)7-CH3Composition is carried out; or consist essentially of (CF)2)3-CH(CH3)-(CH2)2-CH3And CF3-(CF2)3-(CH2)4-CH3And (4) forming.
The composition as defined above is preferably formulated to have a dynamic viscosity, measured at standard ambient temperature and pressure (25 ℃,1 atm), of not more than 10 mPa-s, and preferably not more than 4 mPa-s. Preferably, the composition has a dynamic viscosity of 1 to 4 mPa-s. The viscosity of the composition can be determined using any standard viscometer apparatus known in the art, for example using a glass tube or capillary viscometer.
Described herein as having the general formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3(wherein n is an integer selected from 3 to 5 and m is an integer selected from 1 to 5) and compositions thereof are useful for medical applications, particularly in ophthalmology, particularly topical administration to the eye, e.g., to the lacrimal sac, within the lower eyelid, to the surface of the eye, or to any ocular tissue or anatomical structure associated with the eyeball that may be used for topical administration.
In particular, the compounds of the present invention are useful in the treatment of diseases and conditions that would benefit from the stabilization of the tear film and tear film lipid layer and the lubrication of the ocular surface. Thus, the compositions of the present invention are particularly useful in the treatment of dry eye disease (keratoconjunctivitis sicca) and/or Meibomian Gland Dysfunction (MGD) and any symptom thereof or any symptom associated therewith.
Dry eye, also known as keratoconjunctivitis sicca, can be divided into two categories, namely water-deficient dry eye and evaporative dry eye. These conditions are not necessarily mutually exclusive. Water-deficient dry eye is commonly observed in patients with sjogren's syndrome or patients with lacrimal insufficiency, lacrimal obstruction, or reflex hyposecretion. Evaporative dry eye, on the other hand, has a different root cause and is associated with increased/abnormal evaporative loss of the tear film, for example due to meibomian gland disease, eyelid aperture disease, blinking disease or ocular surface disease.
Symptoms of dry eye include dry eyes, itching, gritty or foreign body sensation; pain, soreness, stinging or burning; itching, increased demand for blinking eyes, eye fatigue, photophobia, blurred vision, redness and inflammation of eye tissues, excessive mucus discharge and crusting/coagulation, contact lens intolerance, and excessive reflex tearing.
Meibomian Gland Dysfunction (MGD) refers to a condition in which the meibomian glands are unable to secrete sufficient oil, or the oil secretion is of poor or abnormal quality. Typically, the oil gland opening may become blocked and clogged, resulting in less oil being secreted by the gland. The oil secreted from the glands may be granular (crusty) or otherwise abnormal, and may irritate the eyes. In the early stages, patients are often asymptomatic, but MGD can cause or exacerbate dry eye symptoms and blepharitis if not treated promptly. The oil glands become clogged due to thickening of the secretions. The chronically obstructed glands eventually become unable to secrete oil, which may lead to permanent changes in the tear film and dry eyes.
Symptoms of meibomian gland dysfunction include dryness, burning pain, itching, sliminess/crusting, tearing (watering), light sensitivity, red eye, foreign body sensation, shot/hordeolum or intermittent blurred vision.
In a preferred embodiment of the present invention, the compounds and compositions thereof as described above are used for topical ocular treatment of evaporative dry eye and/or meibomian gland dysfunction and to alleviate any one of the symptoms associated therewith.
In one embodiment of the present invention, the ophthalmic composition comprises a compound of the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3A semifluorinated compound characterized, wherein n is an integer selected from 3 to 5, m is an integer selected from 1 to 5; or preferably, wherein n is 5 and m is 5 (i.e., formula CF)3-(CF2)5-CH(CH3)-(CH2)5-CH3) Or wherein n is 3 and m is 2 (i.e., formula CF)3-(CF2)3-CH(CH3)-(CH2)2-CH3) For use in the treatment of dry eye disease (keratoconjunctivitis sicca) or symptoms associated therewith. In another embodiment, such compositions may be used to treat meibomian gland dysfunction or symptoms associated therewith.
In a further embodiment, the treatment of these conditions is preferably carried out by a method of administering to a patient in need thereof an effective amount of a composition consisting essentially of said semifluorinated alkane and optionally one or more lipophilic liquid components, preferably essentially non-water-soluble and/or non-water-soluble excipients, for example oily excipients such as lipids, triglyceride oils and any other oil physiologically tolerated by the eye, or other semifluorinated alkanes, such as belonging to the general formula CF3(CF2)n(CH2)mCH3Wherein n and m are independently selected from integers of 3 to 8, e.g. CF3(CF2)3(CH2)4CH3Or CF3(CF2)5(CH2)7CH3。
In a further preferred embodiment of the invention, the compounds and compositions thereof as described above are used for the topical ocular treatment of corneal lesions. Thus, the compounds and compositions are actively supporting the corneal healing process of corneal injury (e.g., corneal erosion).
The advantages of the above compounds in the context of use according to the invention are believed to relate to their properties which are particularly suitable for ophthalmic applications. The close proximity of the refractive index of the compounds of the present invention to that of water means that there will be no or minimal effect on the vision of the patient after administration, unlike ophthalmic compositions based on oily vehicles which may cause blurred vision upon administration. The generally low viscosity and low surface tension of these compounds and in particular their high wetting and spreading ability also ensure that they are quickly contained and adapted for administration on the surface of the eye.
As will be more clearly shown in the examples below, these compounds and compositions thereof were found to be biocompatible and did not exhibit significant cytotoxic effects. Furthermore, the compounds and compositions thereof have been shown to not only have good tolerability in the eye, but also to provide beneficial effects in ocular lubrication and tear film stabilization in the form of relief of symptoms in patients with mild to moderate symptoms associated with dry eye and/or meibomian gland dysfunction. Patients with dry eye and/or dysfunctional meibomian glands often express opaque and thick meibomian plates, which may result in an abnormal lipid layer in the tear film. Without wishing to be bound by theory, it is believed that the physicochemical properties of the compounds of the invention may play a role in stabilizing the lipid layer of the tear film, for example by solubilizing certain lipid components or improving the fluidity of the lipid layer.
In another aspect, the present invention provides a method of treating dry eye and any symptoms or conditions associated therewith, comprising topically applying a composition of the present invention to the lacrimal sac, into the lower eyelid, to the surface of the eye, or to ocular tissue. Preferably, the composition may be administered to the eye or eye tissue up to four times per day.
Furthermore, the present invention provides a kit comprising any one of the compositions as described above and a container for containing the composition. The container preferably comprises a dispensing means, such as an eye dropper, suitable for topical application of the composition to the eye capsule, lower eyelid to the eye or eye tissue.
In a further preferred embodiment, the dispensing means comprises a dropper of a size such that droplets of a volume of about 8 μ L to 15 μ L, preferably a volume of about 8 μ L to 12 μ L, more preferably a volume of about 10 μ L, are dispensed. With small droplet volumes, precise dosing to the eye can be achieved and excessive expulsion of a substantial portion of the composition from the eye after administration can be avoided.
An ophthalmic composition comprising a compound characterized by the general formula:
CF3-(CF2)n-CH(CH3)-(CH2)m-CH3
wherein n is an integer selected from 3 to 5, and m is an integer selected from 1 to 5.
The composition of item 1, wherein n is 5 and m is 5, or wherein n is 3 and m is 2.
The composition according to any of items 1 or 2, which is in liquid form and comprises at least 1 wt% of the compound, in particular from 1 wt% to 5 wt% of the compound, based on the total weight of the composition.
The composition of any one of the preceding claims, formulated as a clear liquid solution.
The composition of any one of the preceding claims, substantially free of:
(a) a polymer,
(b) perfluorinated compounds, and/or
(c) A dissolved pharmacologically active ingredient that is not a semifluorinated alkane.
Composition according to any one of the preceding claims, having a dynamic viscosity at ambient temperature and pressure not exceeding 10 mPa-s, and preferably not exceeding 4 mPa-s.
The composition of any one of the preceding claims, consisting essentially of the compound and optionally one or more lipophilic liquid components.
The composition according to any one of the preceding claims for use as a medicament.
The composition of item 9, item 8, wherein the composition is administered topically within the lacrimal sac, within the lower eyelid, the surface of the eye, or ocular tissue.
Item 10. the composition according to item 8 or 9 for use in the treatment of dry eye disease (keratoconjunctivitis sicca) and/or meibomian gland dysfunction or symptoms or conditions associated therewith.
The composition according to any one of claims 8 to 10 for use in the treatment of corneal injury.
A kit comprising the ophthalmic composition of any one of items 1 to 7 and a container for holding the composition, wherein the container comprises a dispensing device suitable for topical application of the composition to the lacrimal sac, into the lower eyelid, to the surface of the eye, or to ocular tissue.
Item 13. the kit of item 12, wherein the dispensing device comprises a dropper size to dispense droplets having a volume of 8 to 15 μ Ι _.
Examples
CF3-(CF2)5-CH(CH3)-(CH2)5-CH3(2-perfluorohexyl-octane, C14F13H17) Preparation of
Compound CF3-(CF2)5-CH(CH3)-(CH2)5-CH3Can be prepared as follows: free radical addition of perfluorohexyl iodide to 1-octene in the presence of a free radical initiator (herein perfluorohexyl iodide is mixed with 1-octene and a free radical initiator that is AIBN and the resulting solution is held at 80 ℃ for 30 minutes and cooled) followed by hydride (i.e. LiAlH)4) Or by reduction of the resulting iodine adduct by hydrogenation (i.e. catalytic hydrogenation in the presence of a catalyst such as Pd/C) to form 2-perfluorohexyl-octane, followed by purification by fractional distillation. Formula CF as defined above3-(CF2)n-CH(CH3)-(CH2)m-CH3Can be prepared analogously by this general method.
CF3-(CF2)5-CH(CH3)-(CH2)5-CH3:1H-NMR(CDCl3,400MHz):2.17-2.33(m,1H,CH),1.67-1.77(m,2H,CH2),1.25-1.40(m,8H,CH2),1.15(d,3H,CH3),0.90(t,3H,CH3)。
CF3-(CF2)3-CH(CH3)-(CH2)2-CH3(2-Perfluorobutylpentane, C)9F9H11) Preparation of
Compound CF3-(CF2)3-CH(CH3)-(CH2)2-CH3May be prepared according to the general procedure described above using perfluorobutyl iodide and 1-pentene as starting materials.
CF3-(CF2)3-CH(CH3)-(CH2)2-CH3:1H-NMR(CDCl3,400MHz):2.21-2.32(m,1H,CH),1.68-1.74(m,2H,CH2),1.45-1.55(m,2H,CH2),1.12(d,3H,CH3),0.92(t,3H,CH3)。
In vitro cytotoxicity assay
Evaluation of inclusion of 1.3 wt% CF by cell growth inhibition assay3-(CF2)5-CH(CH3)-(CH2)5-CH3And 95.8 weight percent CF3(CF2)5(CH2)7CH3The cell growth inhibition assay predicts a cytotoxic or necrotic effect with good correlation to animal experiments and high sensitivity.
The composition was extracted by cell culture medium (DMEM supplemented with 10% FBS) for about 24 hours with stirring. The resulting extracts were then incubated with mouse cell line L929 cells for 68-72 hours, and then analyzed for protein content using the BCA (bicinchoninic acid) assay as a measure of cytotoxicity. No inhibition of cell growth or cell lysis was observed.
For a catalyst containing about 23.7 wt% CF3-(CF2)5-CH(CH3)-(CH2)5-CH3And about 75.6 wt% F6H8 were similarly fine in vitroAnd (4) determining cytotoxicity.
Tear film analysis study
A composition containing 98.3% by weight CF was tested in an observational study of patients with mild to moderate evaporative dry eye3(CF2)5(CH2)7CH3And 1.2% by weight of CF3-(CF2)5-CH(CH3)-(CH2)5-CH3The composition of (1). The clear colorless liquid composition was provided in a 5ml bottle equipped with a dropper sized to dispense approximately 10 mul of droplets per drop into the eye capsule. The study excluded patients who wear contact lenses. After informed consent, patients were advised to drop 3-4 drops daily for both eyes, with a daily dose of 30-40 μ l following the switch. Patients returned after 5 to 7 weeks of follow-up. Clinical data was collected for 29 patients at baseline and at 5-7 weeks follow-up.
a)Tear film analysis
The tear film fluid and tear film stability improved during the study, as can be seen from the increase in Schirmer I and TFBUT. Retrospective statistical analysis enhanced this observation because differences in TFBUT at baseline and follow-up were very significant (paired two-sided t-test: p ═ 0.0026 (right eye) and p ═ 0.0006 (left eye)). No change in tear osmolarity was detected.
The subjective dry eye questionnaire (ocular surface disease index, OSDI) was shown to contain 98.3 wt% CF3(CF2)5(CH2)7CH3And 1.2% by weight of CF3-(CF2)5-CH(CH3)-(CH2)5-CH3The subjective symptom severity of the patients decreased after a period of 5-7 weeks in the composition of (1), as can be seen by lower scores in follow-up and retrospective statistical analyses (paired two-sided t-test: p)<0.0001)。
b) Cornea staining (oxford grading scheme)
Corneal fluorescein staining is an indicator of corneal injury (loss of cell-to-cell junctions). The data show a reduction in corneal damage after 5-7 weeks of treatment, which can be seen in the number of patients diagnosed with grade 1 or 2 at baseline to the number of patients with grade 0 at follow-up. This difference from the initial lesion level is statistically significant, as shown by the Wilcoxon signed rank test: p ═ 0.0013 (right eye) and p ═ 0.0041 (left eye).
c)Symptom assessment by a physician
At baseline and follow-up the physician asks whether the patient is currently suffering from typical dry eye symptoms. As can be seen in the table below, a lower number of DED-related symptoms were reported after 5-7 weeks of treatment.
d)Meibum secretion analysis
In a healthy eye, meibum is a clear liquid secreted from the meibomian glands. The more opaque and thicker meibum is an indicator of meibomian gland dysfunction. A descriptive examination of the patient's meibum was performed during baseline and follow-up. Based on the data obtained, there were many cases where the eyelid lipid levels were improved. In seven cases, treatment induced a decrease in expressible meibum (from clear meibum to none).
e)Safety parameter
No change in vision or intraocular pressure was observed, indicating the use of the bagContaining 98.3% by weight of CF3(CF2)5(CH2)7CH3And 1.2% by weight of CF3-(CF2)5-CH(CH3)-(CH2)5-CH3The compositions of (a) are safe and do not interfere with these ophthalmic parameters.
Differential scanning calorimetry
Differential scanning calorimetry (DSC 1, Mettler Toledo, Greifensee, Switzerland) for the characterization of CF3(CF2)5-CH(CH3)-(CH2)5-CH3And CF3(CF2)5(CH2)7CH3The structural and phase behavior of the mixture of (a). Data on the transition was obtained by a temperature-increasing scan with a heating rate of 1 ℃/min using DSC. A sealed standard aluminum crucible (40. mu.l, Mettler Toledo) was used.
At CF3(CF2)5-(CH2)7-CH3CF is present in the mixture of (F6H8)3(CF2)5-CH(CH3)-(CH2)5-CH3Resulting in a significant reduction in melting temperature. The enthalpy of fusion is also reduced, indicating that the semifluorinated alkane does not crystallize. This difference would have a beneficial effect on applying the compound to the eye as a tear substitute or lubricant; for example, in terms of its ability to mix with and modulate the tear film lipid layer. In addition, the amount of the compound added to the ophthalmic composition may be variedThis effect is advantageously adjusted.
Also carried out 23.74 wt% of F3(CF2)5-CH(CH3)-(CH2)5-CH3And CF3(CF2)5(CH2)7CH3DSC measurements of a series of mixtures with tetradecane (C14). The transformation data was obtained using temperature-increasing scans (heating rates 0.2 deg.C/min, 0.5 deg.C/min and 1 deg.C/min). The end temperature was determined using a heating rate extrapolated to 0 ℃/minute, while the start temperature was determined using the average from three measurements. With tetradecane and pure CF3(CF2)5(CH2)7CH3A decrease in enthalpy of fusion was observed, indicating that some tetradecane was dissolved in CF3(CF2)5-CH(CH3)-(CH2)5-CH3And the compound is reacted with CF3(CF2)5(CH2)7CH3Compared with the method with stronger solubilizing capability.
Refractive index and evaporation time。
Evaluation of semi-fluorinated alkanes CF3(CF2)3(CH2)4CH3(F4H5) and CF3-(CF2)3-CH(CH3)-(CH2)2-CH3The evaporation time of the mixture of (a). A10. mu.L volume drop of each mixture was placed on a glass surface at room temperature. The time until evaporation was recorded by video monitoring.
Increased amounts of the compound CF were observed3-(CF2)3-CH(CH3)-(CH2)2-CH3It appears that the evaporation time of the mixture is increased (see figure 1).
Similar evaluation of semi-fluorinated alkanes CF3(CF2)5(CH2)7CH3(F6H8) and CF3(CF2)5-CH(CH3)-(CH2)5-CH3The evaporation time of the mixture of (a).
In contrast, the semifluorinated alkanes CF were unexpectedly observed3(CF2)5-CH(CH3)-(CH2)5-CH3The percentage increase of (a) significantly reduced the evaporation time of the mixture (see figure 1).
Thus, depending on the general formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3The compounds characterized, wherein n is an integer selected from 3 to 5, m is an integer selected from 1 to 5, and the amounts present in the mixture, can be adjusted and fine-tuned to the requirements of the intended ophthalmic use, such as extending or decreasing the residence time of the composition on the surface of the eye.
The refractive index of the mixture was also measured. For topically applied ophthalmic compositions, the refractive index of the composition should preferably be similar to or adjusted to that of the eye or lens, e.g., as close as possible to that of physiological tear fluid. If the refractive indices of the compositions are dissimilar, the patient may experience blur or impaired vision when applied to the surface of the eye. Compound CF was observed3(CF2)5-CH(CH3)-(CH2)5-CH3The amount of or the refractive index. FIG. 2 depicts the increase in refractive index values with increasing content of 2-perfluorohexyloctane in a mixture with 1-perfluorohexyloctane. FIG. 3 also depicts the increase in refractive index values as the content of 2-perfluorobutylpentane in a mixture with 1-perfluorobutylpentane increases.
By varying the amount of these compounds in the mixture, e.g. in the formula CF3-(CF2)n-CH(CH3)-(CH2)m-CH3Amount of the characterized compound, wherein nIs an integer selected from 3 to 5, and m is selected from 1 to 5, it is also possible to adjust the composition to the requirements of the intended ophthalmic use, for example to patients with altered tear composition and refractive index due to eye condition and/or age.
Ex Vivo Eye Irritation Test (EVEIT)
For containing CF3(CF2)5(CH2)7CH3Of a semifluorinated alkane, namely CF3(CF2)5(CH2)7CH3And CF3(CF2)5-CH(CH3)-(CH2)5-CH3A composition consisting of a mixture of (having a CF of 0.17% by weight)3(CF2)5-CH(CH3)-(CH2)5-CH3Composition A and having a CF of 64% by weight3(CF2)5-CH(CH3)-(CH2)5-CH3) Composition B) of (1), using hyaluronic acidAs reference, 0.01% BAC (benzalkonium chloride) was used as positive control, with the same as that used in m.frentz et al, alteran.to lab.anim.,2008(36) p 25-32; and an ex vivo eye stimulation assay (EVEIT) similar to that described in Schrage et al, Graefes Arch Clin Exp Ophthalmol 2012(250), 1330) -1340).
A method. Rabbit corneas were obtained and placed in artificial anterior chamber of the eye, which was gently filled with serum-free minimal essential medium (Eagle's MEM) containing Earle's salts and HEPES buffer for nutrition. The medium is continuously replenished by a micro-pump to mimic the physiological condition of the eye. The culture chamber was kept at 32 ℃ in normal air without supplemental CO2And>relative humidity of 95%. In addition to positive controls testing two corneas (n-2), five corneas (n-5)/test substance were used.
After 12 hours of stabilization in the culture chamber, the cornea was evaluated by microscopy and a cornea with intact epithelium and no haze was selected. By usingThe corneal drill applies four small abrasions (2.3-4.3 mm) on the surface of the selected cornea2). All defects were monitored by sodium fluorescein staining (0.17% in water) and microscopy.
The test substance was administered 1 hour after induction of corneal erosion and six times per day to the apex of the cornea (30-50 μ L per four hours). A soft top cannula with continuous suction was placed in the culture chamber at the lowest portion of the corneal scleral area to remove any excess fluid. The experiment was terminated after 3 days of application. Biomicroscopic images of the cornea were taken daily using a phase contrast microscope integrated camera (KY-F1030U, JVC, (BadVilbel, DE) mounted on a Z16 APO microscope (Wetzlar, DE) to record the corneal healing process. All defects were monitored by sodium fluorescein staining (0.17% in water) and yellow-green fluorescence indicated areas of epithelial defects. Erosion size was determined using a software tool of a microscope (disckus). At the end of 3 days, the experiment was terminated and all corneas were fixed in 3.7% formaldehyde and stained with hematoxylin-eosin dye for microscopic evaluation. To monitor the metabolic activity of the cornea, glucose and lactate concentrations were quantified photometrically in the outflow medium from the artificial anterior chamber.
And (6) obtaining the result. Hyaluronic acid composition with standard reference substanceIn contrast, after induction of corneal erosion, a similar positive effect of the two semifluorinated alkane mixtures (compositions a and B as described above) was observed in the corneal therapeutic process.
Corneal erosion size measurement/mean mm2(SD)
Histological observations on day 3
In a composition comprising 64% by weight of semifluorinated alkane CF relative to the total weight of the composition3(CF2)5-CH(CH3)-(CH2)5-CH3No significant difference in positive corneal healing was observed between composition B and composition a of (a). For both compositions, as with the reference composition, a significant reduction in mechanically induced epithelial erosion was found and was essentially absent after day 2 of treatment. Figure 3 depicts corneal erosion size measurements of test compositions, references and positive controls for day 0-3 of the EVEIT experiment.
As shown in the above table, the microscopic histological examination of the cross section of the cornea after the end of the experiment on day 3 revealed that the composition A, B and the reference were usedThere were no significant residual defects or differences in the treated cornea.
Furthermore, no corneal toxicity based on metabolic activity as indicated by glucose/lactate measurements was observed for these compositions.
In sharp contrast, a positive control containing 0.01% preservative BAC was observed, inducing a gradual increase in epithelial damage over the course of three days of the experiment.