WO2024239103A1

WO2024239103A1 - Topical cream for treating dermatological conditions

Info

Publication number: WO2024239103A1
Application number: PCT/CA2024/050659
Authority: WO
Inventors: Marc Purcell
Original assignee: Bio A3P Inc.
Priority date: 2023-05-19
Filing date: 2024-05-17
Publication date: 2024-11-28

Abstract

There is provided a topical cream for treating, preventing or alleviating the symptoms of a dermatological condition requiring energy supplementation by adenosine triphosphate (ATP). The topical cream includes, as an active ingredient, chloroplasts having functional thylakoid membranes and a gel carrier. The topical cream is light activated.

Description

TOPICAL CREAM FOR TREATING DERMATOLOGICAL CONDITIONS

TECHNICAL FIELD

[0001] This disclosure relates to the field of topical creams used in the treatment of dermatological conditions including itching, inflammation, pigmentation and non-cancerous growths.

BACKGROUND OF THE ART

[0002] The treatment of certain dermatological conditions with topical creams has not provided efficacious and consistent results with the topical creams currently available. It would be desirable to provide an efficacious topical cream for the treatment of acute or chronic skin conditions such as eczema, with minimal or negligible side effects. In particular, topical creams containing natural ingredients only is a preferred option for consumers. Accordingly, improvements in the efficacy of topical creams in treating dermatological conditions is sought.

SUMMARY

[0003] There is provided a method of treating, preventing or alleviating the symptoms of a dermatological condition requiring energy supplementation by ATP by applying on the skin of a subject in need thereof, a topical cream comprising, as an active ingredient, chloroplasts having functional thylakoid membranes and a gel carrier, and illuminating the topical cream with light. There is also provided the use of the topical cream in the treatment, alleviation of symptoms or prevention of dermatological conditions requiring energy supplementation by ATP. Preferably the topical cream comprises from 0.25 to 3 wt. % of a lyophilised plant extract containing chloroplasts having functional thylakoid membranes, from 67 to 73 wt. % of water, from 4 to 6 wt. % of oils, from 1 to 3 wt. % of essential oils, from 0.5 to 1 .5 wt. % of a gelling agent, from 3 to 5 wt. % of an antimicrobial agent, and from 3 to 7 wt. % of an emulsion agent. Optionally, the topical cream further comprises one or more of 4 to 8 wt. % of an emollient, from 0.1 to 0.3 wt. % of an antioxidant, 0.5 to 1 .5 wt. % of a cooling agent, 0.5 to 1 .5 wt. % of a preservative, and from 0.25 to 1 wt. % of a ultra-violet (UV) protection agent. The dermatological condition is preferably selected from redness, itchiness, dryness, irregular skin coloration, milium, firmness, brown spots, wrinkles, rashes, keratosis, inflammation, pigmentation, non-cancerous growths, lesions, eczema and combinations thereof. [0004] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.

DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1 is a graph showing the relative adenosine triphosphate (ATP) production in function of luminosity.

[0006] FIG. 2A is a graph showing the relative ATP production at different time points of alternating light and dark conditions.

[0007] FIG. 2B is a microscopy image showing re-suspended lyophilized chloroplasts.

[0008] FIG. 3A is a photograph of an arm of a subject with eczema.

[0009] FIG. 3B is a photograph of the arm of Fig. 3A after treatment with a topical cream according to the present disclosure.

[0010] FIG. 4A is a photograph of the skin of a subject with keratosis and dark spots.

[0011] FIG. 4B is a photograph of the skin of Fig. 4A after treatment with a topical cream according to the present disclosure.

[0012] FIG. 5A is a photograph of the skin of another subject with keratosis and dark spots.

[0013] FIG. 5B is a photograph of the skin of Fig. 5A after treatment with a topical cream according to the present disclosure.

[0014] FIG. 6A is a photograph of the skin of a subject with keratosis.

[0015] FIG. 6B is a photograph of the skin of Fig. 6A after treatment with a topical cream according to the present disclosure.

[0016] FIG. 7A is a photograph of the facial skin of a subject with lesions and redness.

[0017] FIG. 7B is a photograph of the skin of Fig. 7A after treatment with a topical cream according to the present disclosure.

DETAILED DESCRIPTION [0018] There is provided a topical cream for the treatment of dermatological conditions, the topical cream comprising chloroplasts having functional thylakoid membranes able to produce ATP in the presence of light and a gel carrier. The chloroplasts are the active ingredient in the topical cream through their ability to produce ATP by photosynthesis. An advantage of the topical cream of the present disclosure is that when it is applied to the skin of a subject and exposed to light the ATP production occurs directly on the skin of the subject and enters the epidermis to provide beneficial effects. The dermatological conditions considered by the present disclosure include conditions where energy supplementation by ATP would provide a beneficial effect such as a reduction of symptoms, a treatment of the condition and/or a prevention of the condition. The topical cream targets the treatment, the prevention or the alleviation of symptoms for at least one of redness, itchiness, dryness, irregular skin coloration, milium (commonly known as milk spots), firmness, brown spots, wrinkles, inflammation, pigmentation, non-cancerous growths, rashes, keratosis, lesions and eczema.

[0019] The chloroplasts included in the topical cream are lyophilized chloroplasts (e.g. present in a plant extract lyophilized powder) that have been re-suspended into the topical cream. In some embodiments, the plant extract powder added in the topical cream is present in a concentration of 0.1 to 10 wt.%, 0.25 to 10 wt. %, 0.25 wt.% to 5 wt. %, 0.25 to 3 wt. %, preferably from 0.5 wt. % to 2 wt. %, with respect to the total weight of the cream. The concentration is below 10 wt. % because at high concentrations of chlorophylls the topical cream may stain the clothes of the subject applying the topical cream on their skin. Moreover, as will be explained in more detail below, the production of ATP does not only depend on the concentration of chlorophylls included but also on the exposure time and intensity of illumination. Thus, lower concentrations of the plant extract such as at least 0.1 wt. % or at least 0.25 wt. % are sufficient to provide adequate ATP production with sufficient exposure time and intensity of illumination. In some embodiments, the plant extract in a concentration of at least 0.1 wt. % or at least 0.25 wt.% the fluorescent activity quantified as Fv/Fm (see Example section) is more than 0.1 , preferably more than 0.15, more preferably more than 0.2. In some embodiments, the chloroplasts make up at least 50 wt. %, at least 70 wt. %, at least 80 wt. %, at least 90 wt. %, at least 95 wt. %, at least 97 wt. % or at least 98 wt. % of the plant extract. In some embodiments, the resulting total chlorophylls concentration (note that the chlorophylls are contained in the chloroplasts) in the topical cream can be from 1 mg/mL to 50 mg/mL, particularly, from 1 mg/mL to 40 mg/mL, and still particularly from 15 to 50 mg/mL. In some embodiments, the remaining water content in the plant extract that has been lyophilized is less than 10 wt. %, preferably less than 7 wt. %, preferably less than 5 wt. % and most preferably less than 3 wt. %.

[0020] In exemplary embodiments, the topical cream comprises from 0.25 to 10 wt. % of the lyophilised plant extract containing chloroplasts having functional thylakoid membranes, from 67 to 73 wt. % of water, from 4 to 6 wt. % of oils, from 1 to 3 wt. % of essential oils, from 0.5 to 1 .5 wt. % of a gelling agent (preferably a polysaccharide gelling agent such as Amigel™), from 3 to 5 wt. % of an antimicrobial agent, optionally from 3 to 5 wt. % of a hydration agent such as glycerin, from 3 to 7 wt. % of an emulsion agent, optionally from 4 to 8 wt. % of an emollient, optionally from 0.1 to 0.3 wt. % of an antioxidant, optionally from 0.5 to 1.5 wt. % of a cooling agent, optionally from 0.5 to 1 .5 wt. % of a preservative, and optionally from 0.25 to 1 wt. % of a ultraviolet (UV) protection agent. In some embodiments, the oils are safflower oil and/or argan oil. In some embodiments, the essential oils are spearmint and/or rosemary oil.

[0021] Chloroplasts are membranous organelles that serve as the site of photosynthesis, and they have a major structural and functional importance for the present invention. Typically, chloroplasts comprise three types of membranes, which are: (i) a smooth outer membrane, which is freely permeable to molecules; (ii) a smooth inner membrane, which contains many transporter proteins such as integral membrane proteins regulating the exchange of small molecules like sugars and proteins between the cytoplasm and the chloroplast; and (iii) a system of thylakoid membranes which contains the chlorophyll (types a, b, and c) and carotenoids which are sites of the light-dependent reactions. Particularly, chlorophyll a plays a central role in converting light energy into chemical energy. Other accessory pigments are also part of the structure of chloroplasts such as carotenoids and xanthophylls. These are involved in getting rid of excess absorbed energy as heat. In order to protect the chloroplasts from degradation and to avoid the ATP production priorto topical administration, the topical cream of the present disclosure is stored in darkness.

[0022] The plant extracts referred to herein in the present disclosure can be extracted from different plant species and different corresponding methods, as long as the extract contains chloroplasts. The plant species including but are not limited to cyanobacteria, algae, bryophyta, and vascular plants. Preferably, the plant extract is obtained from spinach, more specifically spinach leaves. The plant extracts can be generally obtained by a homogenization that includes grinding at least a portion of the plant that contains the chloroplasts (for example the leaves). The ground plant is homogenized in a buffer having a pH of from 4 to 9 and a viscosity of 2-10 cP at a temperature between 0 and 40 °C and preferably under 4 °C to avoid any degradation of the thylakoid membranes. If homogenization is performed in deionized water or another medium that does not have the appropriate viscosity and osmolarity the thylakoid membranes may shrink or burst and therefore disintegrate either at the homogenization step or in the subsequent steps particularly the lyophilisation. Moreover, performing lyophilisation directly on the plant extract without any liquid medium added would also disintegrate the thylakoid membranes. In preferred embodiments, the buffer comprises a sugar (e.g. sucrose) and a salt (e.g. sodium chloride). The role of the buffer is to control the acidity and osmolarity of the solution and to maintain the pH in the ranges described herein. In some embodiments, the buffer is a Tris buffer, N-2- hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) buffer or phosphate buffered saline (PBS). In preferred embodiments, the pH is from 7 to 8 or more particularly 7.4. The homogenization buffer is preferably included in a ratio of plant (g) to buffer (mL) of from % to 1/3.

[0023] Following the homogenization, the homogenate obtained was subjected to a separation step to separate out cell debris. This can be performed with many different methods for example a centrifugation system can perform a separation by size. The centrifuge can be provided with a 70 pm filter through which the homogenates are passed, but on which cell debris are retained. Following the separation step a resuspension is performed using a buffer having a composition as described for the homogenization buffer. In one example, the resuspension buffer is a phosphate buffer saline (PBS) containing 0.175 M sucrose and 0.01 M sodium chloride and having a pH of 7.4. The resuspension is then subjected to lyophilization at temperatures preferably below -12 °C, such as in the range of from -16 to -22 °C, more particularly -18 °C. The resulting lyophilized powder can be stored under freezing temperature of from -30 to -40 °C for example. The plant extracts of the present disclosure can therefore be free of cell debris.

[0024] It is believed that the production of ATP directly at the site of the dermatological condition provides the treatment benefits described herein. ATP or adenosine tri-phosphate with the chemical formula C10H16N5O13P3 is composed of a nitrogenous base (adenine), a sugar with 5 carbon atoms (deoxyribose) and three phosphate groups. The bond between the second and third phosphate groups releases energy.

[0025] Photosynthesis is known as the principal process converting light energy to biochemical energy. Chloroplasts are composed of a lipid bilayer membrane (thylakoid) arranged in nanodiscs (grana). Thylakoidal membranes are composed of light absorbing molecules called pigments capable of absorbing photons at specific wavelengths (A) and reflecting others. Chlorophyll in its two forms a and b represents the main pigment of thylakoidal membranes. Especially chlorophyll a plays a central role in converting light energy to chemical energy. Other accessory pigments are also part of the structure of chloroplasts such as carotenoids and xanthophylls. They are involved in getting rid of excess absorbed energy as heat.

[0026] ATP synthase also called F0F1-ATP synthase is a large molecular motor enzyme complex playing a central role in the generation of cell’s energy. This protein complex structure comprises two main parts (domains): Fo and Fi. Fi roots in the stroma and F_o is set in the membrane. F_o is a cylindrical structure embedded into the thylakoid membrane and is linked to a central rod that allows it to turn into Fi. F_o domain of ATPase is capable of rotation when driven by translocated protons H⁺. The proton gradient across the membrane drives F_o to rotate and powered the synthesis of ATP from ADP and inorganic phosphate (Pi) in its Fi domain. The chloroplast has an electric potential (Aqj) and a pH gradient (ApH) created across the thylakoid membrane. The pH gradient (also referred to as a proton gradient) drives the synthesis of ATP by the transmembrane protein ATP synthase which includes domains Fo and Fi. The electrochemical proton gradient drives the rotation of the membrane-embedded Fo domain which powers the synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate in the Fi domain. Intact chloroplasts are essential to preserve the contents of the stroma where Fi-ATP synthase subunit protrudes, and ATP energy is released. The movement of electrons and protons H⁺ across the thylakoid membrane generates an electric potential (Aqj) and a chemical proton gradient (ApH). The two forces highly interwoven work in conjunction to create an electrochemical gradient or driving force of the proton (proton motive force, pmf) across the thylakoid membrane. The dynamics of the electric potential (Aq ) and pH gradient (ApH) across the thylakoid membrane can be affected by the electrochemical nature of the products used (product added) in the topical cream formulation. It was presently found that the addition of compounds that contain groups accepting protons contributes to the dissipation or even destabilization of transmembrane proton flow driven by a pH gradient hence forcing F0F1-ATP synthase to stop, take a break, go in a rest state or reduce its activity. Particularly, Hymagic™ 4D and hydrolyzed jojoba esters (HJE) were found to be detrimental to the production of ATP by the topical cream.

[0027] Some hydrophobic molecules may have the ability to tether into the thylakoid membrane and increase its permeability to ions either as transmembrane transporters or by forming an ion channel. These molecules bind to thylakoid membranes through their hydrophobic chain while their hydrophilic part facilitates the diffusion of ions across the membrane and an ionophoric mechanism (protonophoric when it involves protons H⁺ transport) arises as a result. The transport takes place according to a chemiosmotic concentration gradient, i.e., from the most concentrated medium to the least concentrated. In some embodiments, the ingredients for the composition are such that the pH gradient achieved across the thylakoid membrane of the chloroplasts is maintained in the topical composition to be similar to a physiological pH gradient. Therefore, in some embodiments, the topical cream is free of Hymagic™ 4D and HJE.

[0028] Unlike the proton gradient related to the rotary motion of Fo-ATPase which is coupled to an energy source (electronic potential induced by electron transport), the proton gradient induced by protonophores is a passive transport that requires no energy source and when present it can interrupt and rest the pH gradient involved in the activation of ATPase. The shortage of a single potential (electrical or proton) affects ADP phosphorylation.

[0029] The rotary movement of the Fo-ATP synthase subunit which ensures the driving of protonation from the inside and deprotonation to the outside is destabilized decreasing the ATP production. If there is no proton reflux, the Fo-membrane rotor of the enzymatic complex cannot be operated.

[0030] ATP is synthesized by coupling two reactions, electron transport and pH gradient. These 'protonophore' molecules act like uncoupler agent by mimicking its mechanism of action they short-circuit the electronic potential and the pH gradient and break the link between the two creating a gap in the energy transport process essential for the activation of ATPase. Hacking the proton motive force of pH gradient, these compounds impede the oxidative phosphorylation of ADP to ATP and puts the F0F1-ATP synthase at rest.

[0031] The resulting gap between the Aqj and ApH ratio slows the proton motive force and creates a back-pressure on photochemical reactions because of the lack of ADP phosphorylation. The energy produced by redox reactions cannot be used for phosphorylation of ADP to ATP.

[0032] Uncouplers act as catalysts and not as specific inhibitors, thus in the presence of a protonophore faking uncoupler, ATP synthesis cannot take place even though the activities of electron flow and ATPase are not inhibited, but at rest. An instability of electrocharges (positiveprotons, negative-electrons) disrupts the electromagnetic field and the pumping of H+ protons across the thylakoid membrane, which goes against the driving force of protons generating ATP synthesis. Directly or indirectly these disturbances in electrocharges distribution affect the acidification or alkalinization of the environment and cause an imbalance of the Aqj/ApH ratio. [0033] The product’s flow resistance force or viscous force regarding the diffusion of the molecules involved in the energy production process, is proportional to the density of the product. Dynamic viscosity and density of the product, which thus define its kinematic viscosity, create resistance forces around the chloroplast’s particles. This slowdown/stoppage produces a direct effect on the movement and diffusion of the molecules carrying the energy necessary to start the rotary motion of Fo-ATP synthase. Hymagic4D, for example, has a high crosslinking density that decreases its fluidity and increases the flow’s resistance of the product. This characteristic makes it incompatible with the topical cream of the present disclosure since the active particles of our extract must conserve a certain fluidity in their membrane and in their movement.

[0034] Among uncouplers we can name phenols, benzimidazoles, N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines. In preferred embodiments, the topical cream is free of any uncoupler.

[0035] One particular molecules acting similar to an uncoupler that was identified are the hydrolyzed jojoba esters (HJE). This weak alkaline pH compound is likely to capture one or more H⁺ protons. HJE is a mixture of fatty acid and fatty alcohol. With its dual amphiphilic character, this molecule is able to tether to the thylakoidal membrane via its hydrophobic chain, while the polar head (carboxylate group -COO- for fatty acids and hydroxylate -O- for fatty alcohols) gives the molecule its ability to capture an H⁺ proton. Freeing itself from its highly ordered structure, HJE allows H⁺ protons to take a side road to cross between lumen and stroma, thus interrupting the protons gradient across Fo-ATP synthase. The proton gradient through the Fo-ATPase subunit and ATP synthesis do not operate separately, they are in coupling, i.e. one conditions the other.

[0036] The couples (-COOH/COO-) consisting of the acid form of fatty acid and its conjugated base thus serve as a shuttle of protons H+ across the thylakoid’s membrane. Also, the strong polarization of the O-H bond of fatty alcohols and the weak acid character which gives them the possibility of ionic rupture (release of the H + cation of the OH group) predisposes them to transmembrane proton exchanges. These reactions are therefore reversible and both molecules can act as both donors and acceptors of H+ protons.

[0037] Unlike the protonophoric effect mentioned above, other compounds suspended in the bathing medium can act as electrolytes capable of affecting the electric field across the thylakoid membrane and electrolyte nature of the luminous matrix. [0038] Polar group compounds can easily ionize and release (or accept) H⁺ protons which generates acidification (or alkalization) of the lumen and leads to activation (or dissipation) of the pH gradient. High molecular weight hyaluronic acid is richer in electrons and therefore more easily oxidizable. Hyaluronic acid carries a lot of negative charges in its molecular structure and can bind to water molecules.

[0039] In some embodiments, the electrical potential difference across the thylakoid membrane is also maintained to be similar to the physiological potential difference. However, the electrical potential difference has an indirect and less significant effect on the ATP production compared to the effect of the pH gradient as will be explained below.

[0040] Pigments acting as photoreceptors form in conjunction with proteins two multi-subunit protein-membrane complexes called photosystems. There are two photosystems, PSII and PSI each of them consisting of an antenna complex collecting energy (LHC) and a reaction center with maximum absorption at wavelengths 700 and 680 nm for PSI and PSII, respectively. Powered by light, the two complexes PSII and PSI work in series to convert light energy into biochemical energy. These pigment-protein complexes capture photons of light through their lightharvesting pigments complexes (LHC) and initiate a series of redox reactions (transfer of electrons through specialized molecules such as plastoquinones (PQ)).

[0041] Acting as a master on switch, the light energy of particles (photons) of electromagnetic radiation captured at the LHC complexes is transported to the reaction centers of the photosystem II (PSIls) where a special molecule of chlorophyll P680 is excited by passing to its oxidized state P680+ and releasing 1 electron which will be transferred through a series of redox molecules. In PS II, photolysis of water occurs at the oxygen evolving complex (OEC) in order to replace the electrons released by PS II. The OEC oxidizes two molecules of water releasing 1 molecule of oxygen, 4 electrons and 4 protons H⁺ as in the reaction below:

2 H₂O -► O₂ +4 H⁺ + 4 e-

[0042] During the electron transport chain, PS II passes electrons to plastoquinone (PQ) molecules which carries them to the complex cytochrome b6f (Cytb6f). For each molecule of hydrolyzed water, two molecules of PQ are protonated to form 2 molecules of plastoquinol (PQH2). The overall reaction in PS II is shown below:

2PQ + 2H₂O -► O₂ + 2PQH₂ [0043] The Q cycle is a catalytic mechanism that couples electron transfer through the Cytb6f complex to proton translocation from the stroma to the lumen. The thylakoid membrane is globally impermeable to protons. The movement of electrons and protons H⁺ across the thylakoid membrane generates an electric potential (Aqj) and a chemical proton gradient (ApH). The two forces highly interwoven work in conjunction to create an electrochemical gradient ordriving force of the proton (proton motive force, pmf) across the thylakoid membrane.

[0044] The proton motive force or driving force expressed in volts is defined as:

[0045] where ApH= transthylakoidal pH difference; Aqj = electrical potential difference; I = lumen; s= stroma; R= Universal gas constant; T = temperature and F = Faraday constant.

[0046] This driving force is dominated by ApH after the first 60 seconds of continuous illumination under low and high light conditions. At steady state, the transthylakoidal driving force consists mainly of ApH, the contribution of Aqj to steady state is negligible (< 10%).

[0047] Light-driven electrons causes protons H⁺ to pass from the stroma to the lumen and decrease the pH of the lumen. The molecular motor enzyme ATP synthase facilitate the return of protons to the stroma and simultaneously the synthesis of ATP.

[0048] The transfer of electrons through a series of redox intermediates coupled with proton transfer reactions in the thylakoid membrane create the potential energy for ATP synthesis. Redox reactions involving a transfer of electrons coupled to the pH gradient that engage a movement of H⁺ protons are responsible for the movement and rotation of the ATPase pump.

[0049] Non-photochemical quenching (NPQ) is a mechanism employed by plants and algae to protect themselves from the adverse effects of high light intensity. It involves the quenching of singlet excited state chlorophylls (Chi) via enhanced internal conversion to the ground state (nonradioactive decay), thus harmlessly dissipating excess excitation energy as heat through molecular vibrations. The increase in ApH acts as a trigger for non-photochemical quenching qE. NPQ is mainly composed of qE related to the energization of the thylakoid membrane. qE involves the pH gradient and xanthophyll cycling (conversion of violaxanthin to antheraxanthin and zeaxanthin, by catalytic action of the enzyme violaxanthin deepoxidase). NPQ can be substituted for qE, since qE is a significant fraction of NPQ. [0050] NPQ conduct conformational changes in the LHCII complex which allow light collecting antenna to be turned on and off. As a negative feedback loop, it controls the major collecting antenna complex (LHC) according to the energy requirements of light phase and regulate the frequency of photons capture and electron turnover rate in PSII reaction centers. Recent reports have confirmed that the in vivo site of qE is located in the major trimeric light harvesting complex (LHCII).

[0051] The absence of ApH prevents the formation of qE and consequently blocks the rotary motion of Fo subunit of the ATP synthase which slows down and prevent the phosphorylation of ADP to ATP.

[0052] Electrically charged particles creating a field destabilization or large increases in electric field (Aqj) across the thylakoid membrane induce photosystem II recombination reactions that produce damaging singlet oxygen (102). These changes also induce inconsistencies in the osmotic balance between lumen and stroma.

[0053] In addition, the balance of Aqj/ApH and its kinetics depend on the homeostasis regulation in the chloroplast. It is believed that pmf partitioning into Aqj and ApH can be controlled by regulating the ionic strength and balance of the chloroplast. The ions distribution between lumen and stroma is under control of ion channels/transporters which model the kinetic of A ¹ and fine-tune the pmf. The ion gradient is essential for regulating enzyme activities and transporting cellular energy. In the chloroplast, ions promote the stacking of granas and regulate enzymatic activities including ATPase mediating ATP synthesis.

[0054] An additional benefit of the present topical cream is the absorption of light in the blue range (400-500 nm). Blue light is associated with aging and therefore the topical cream preserves the skin when exposed to blue light. A further benefit of the present disclosure is the consumption of carbon dioxide as part of the photosynthesis performed by the chloroplasts which therefore reduce the amount of carbon dioxide that the skin is exposed to.

EXAMPLE 1

[0055] A plant extract was obtained from leafs of the spinach plant species Spinacia oleracea. first homogenizing the spinach by mechanical grinding. The mesophylium tissues (leaves or needles) and stems were cut into small pieces with a rotative knife. The homogenization was performed under 4 °C to avoid any degradation of the tissue during the procedures. The tissue was homogenized in a homogenization buffer composed of 0.3 M sucrose, 50 mM Tris buffer (pH 7.4) and 10 mM sodium chloride (extract solution). Taking spinach as a reference plant, the wet weight ratio of plant leaf tissues (g) I volume of buffer (ml) was of about 1/2 to 1/3. The plant was mixed with the buffer and homogenized in a commercial blender for about 1 minute. Homogenates were separated from cell debris and soluble components by continuous centrifugation, at about 2000 x g for 5 minutes. The centrifugation system allowed for the isolation of the homogenates based on their size, as the centrifugation was provided with a 70 pm filter through which the homogenates was passed, but on which cell debris were retained. The pellet (chloroplasts) was suspended in homogenization phosphate buffer (pH 7.4) containing 0.175 M sucrose and 0.01 M sodium chloride (re-suspension solution). The suspension was transferred to a container a third of its volume (maximum) and placed in a freezer (-18 °C) until completely frozen. The container is placed in a freeze-dryer (temperature around -34 °C) for 24-48h. The leafs characterization is presented in Table 1. The leafs were ground and then the fine and coarse portions were separated. The fine portion was then subjected to a lyophilization. The composition of the plant extract is summarized in Table 2.

Table 1 . Spinacia oleracea leafs

* The Fv/Fm ratio was calculated as follows:

Fo : Basic fluorescence yield (relative units) recorded with low measuring light intensities.

F_m: Maximum chlorophyll fluorescence yield when photosystem II reaction centers are closed by a strong light pulse (relative units).

F_v / F_m = (F_m - Fo) / F_m; maximum quantum yield of photochemistry of photosystem II

Table 2. Plant extract composition

[0056] A topical cream composition was produced by first mixing under high agitation 50 g of Amigel™ (Alban Muller) (final concentration of 1 wt. %), mineralized water (3.495 kg) (final concentration of 69.895 wt. %) and 1.5 g of sodium hydroxide (final concentration of 0.03 wt. %) to obtain a mixture labeled as “A”. Separately, 187.5 g of glycerin and 15 g of Keltrol™ CG-SFT (respective final concentrations of 3.75 wt. % and 0.3 wt. %) were mixed to obtain a mixture labeled “B”. Mixtures A and B were then mixed for 30 mins under heating at a temperature of from 78 to 80 °C to obtain the mixture “AB”. [0057] A third mixture labeled “C” was produced by mixing under heating at a temperature of from 78 to 80 °C the following components: 250 g of Olivem™ 1000 (Barentz) for a final concentration of 5 wt. %, 150 g of Oliwax™ LC (Barentz) for a final concentration of 3 wt. %, 100 g of safflower oil (New Directions) for a final concentration of 2 wt. %, 100 g of argan oil (New Directions) for a final concentration of 2 wt. %, 250 g of Centiol™ OE (Brentag) for a final oncentration of 5 wt. %, 50 g of Lexfeel™ N-100 (Debro) for a final concentration of 1 wt. %, 10 g of Coviox™ T-70 (Brentag) for a final concentration of 0.2 wt. %, and 50 g of Frescolat™ for a final concentration of 1 wt. %.

[0058] Mixture C was then added to mixture AB and was homogenized for 10 mins then cooled and scraped to obtain mixture “ABC”. At 45 °C, the following components were added to the mixture ABC and mixing was performed in between the addition of each component: 50 g of Plantservative™ Wsr (Azelis) for a final concentration of 1 wt. %, 150 g of Aquacell™ (Barnet) for a final concentration of 3 wt. %, 5 g of mini hyaluronic acid (Azelis) for a final concentration of 0.1 wt. %, and 25 g of Borealine™ Protect (Lucas Meyer Comestics (IFF)) for a final concentration of 0.5 wt. %. The pH was then adjusted to be between 5.75 and 6.25 by the addition of citric acid for acidification and NaOH for basification. Finally, at 35 °C, 25 g of the plant extract containing chloroplasts having functional thylakoid membranes is added to the mixture for a final concentration of 0.5 wt. %. Essential oils are also included, specifically 0.15 wt. % (7.5 mL) of Spearmint and 0.075 wt. % (3.75 mL) of Rosemary essential oils were added to achieve homogeneity. The quantity of essential oils may be modified and adapted to ensure homogeneity.

[0059] The ATP synthesis of the obtained cream was evaluated after exposing the cream to ambient light. It was observed that after exposure to ambient light for one minute the peak of ATP production was reached. The effect of the luminosity was evaluated (Fig. 1) to compare the ATP production obtained at lower luminosity than ambient light. It was observed that at 8000 lux, 80% of the peak ATP production is achieved. The luminosity of an office is generally around 1000 lux accordingly it would be preferred to expose the topical cream to natural light from the sun for optimal performance.

[0060] When the cream is applied on the skin of a subject the subject may be moving from darker to lighter areas of their household. Accordingly, the regeneration and recovery of ATP production was evaluated after exposures to dark conditions. Specifically, at TO the topical cream was in darkness, at T 1 the topical cream was exposed to light for 30 seconds then again darkness for 10 minutes at T2. The periods of light (30 seconds was repeated i.e. T1 , T3,, T5 and T7) interspaced with conditions of darkness for 10 minutes (i.e. T2, T4, and T6). The results are shown in Fig. 2A. It was observed that the production of ATP regenerates upon re-exposure to light after a significant period of darkness of 10 minutes.

[0061] To demonstrate that the thylakoid membrane remained functional after the lyophilization process the lyophilized powder was resuspended in PBS containing 0.175 M sucrose and 0.01 M sodium chloride and having a pH of 7.4 and the chloroplasts were observed using then observed by microscopy. The microscope used was a Leica (DM RB) bright field florescence microscopy. Microscopy confirmed the presence of viable chloroplasts having functional and intact membranes (Fig. 2B).

[0062] The viscosity of the topical cream was analyzed and found to be in the range of 9,000 - 12,000 cP.

[0063] By virtue of performing photosynthesis, the topical cream of the present disclosure can be considered an anti-pollution cream. Indeed, the cream converts carbon dioxide into oxygen. Accordingly, the present disclosure also contemplates herein the use of the cream to reduce the concentration of carbon dioxide as a anti-pollution cream.

EXAMPLE 2

[0064] To determine whether certain compounds that may be present in topical creams act as uncouplers and prevent or reduce the formation of ATP, the ability of chloroplasts in creams to absorb photons of light and generate ATP was measured by the maximum efficiency of the main multi-subunit protein-membrane complex PSII as initiator of photochemical reactions, by using the chlorophyll fluorescence parameter Fv/Fm ratio.

[0065] Chlorophyll fluorescence analysis allows instantaneous measurement of key aspects of light capture and electron transport. To deduce information on photochemical activation of muti subunits complexes PSII and ATP synthase, Fv/Fm ratio is highly valuable tool in understanding and making predictions regarding photochemical activity. Measurements were performed using the modulated fluorometer Junior-PAM (Pulse Amplitude Modulation, Walz, Effeltrich, Germany) which detect and amplify the fluorescence excited by a constant measuring beam and saturated by an actinic saturating pulse. This saturating flashes momentarily close all PS II centers and record the maximum PSII efficiency (Fv/Fm). [0066] A sample of the base cream formula containing the A3P bioactive extract dark-adapted was excited by a saturating pulse and instantaneously the ratio Fv/Fm was recorded. The same measurements were carried out on samples of base cream containing the extract A3P to which was added individually each of the compounds listed in Table 3 in order to test their biocompatibility with our formulation. The percentages presented in the table are a weight percentage with respect to the total mass of the cream composition.

Table 3: Results of the compatibility of various molecules with chloroplasts and ATP production

[0067] HJE and Hymagic™ 4D did not show strong compatibility with the cream formula tested (Table 3). Indeed, light-induced photochemical reactions result from a movement of electrons and protons across the thylakoid membrane that generate an electrical potential and a chemical gradient of protons essential for ATPase activation. The coupling of Aqj and ApH is essential for ATPase activation. HJE appears to act as a fake-uncoupler that breaks the link between the two processes Aqj and ApH by creating a proton scattering channel across the thylakoid membrane. It is emphasized that this protonophoric effect not only stops the ATPase enzyme, but also slows electron traffic on the side of PSII i.e., principal initiator of the photosynthetic machinery, which results in a lower Fv/Fm ratio.

[0068] For its part, high molecular weight hyaluronic acid is known to have too many negative charges which may disrupt the electromagnetic field of chloroplasts and may create an electrolyte effect that also results in a lower Fv/Fm ratio. [0069] The two identified non-compatible components do not deactivate the chloroplastic machinery for both ATPase and photosystems, but they disrupt their operation either by slowing/interrupting the traffic of electrons and protons H+ which together constitute the driving force of protons main activator of the ATPase enzyme and therefore photophosphorylation of ADP to ATP.

[0070] It was therefore shown herein that some compounds can slow down the production of ATP by acting on the main photoreceptor complex and breaking the coupling between the two proton and electron forces, namely ApH and Aqj. Not being classified as traditional uncouplers, these compounds mimic the mechanism of action of a uncoupler and break the link between two coupled reactions that collaborate to produce ATP.

EXAMPLE 3

[0071] To test the efficacy of the topical cream of the present disclosure, the topical cream as obtained from Example 1 was tested on thirty-five subjects. Applications of the cream were performed daily for 3 to 5 weeks. When applied on the skin, the subjects were instructed to expose the cream to light for at least 5 minutes. The skin conditions among the thirty-five subjects included redness, rash, itchiness, dryness, keratosis, brown spots, lesions, and eczema. Table 4 presents the results obtained before and after application of the topical cream of the present disclosure.

Table 4. Treatment results before and after the topical cream

++ = highly developed state, + = attenuation, - = disappearance

[0072] In one case, the subject presented itchiness and eczema that was not cured after 4 years of traditional treatments. The traditional treatments were the administration of treatment that contain cortisone and clinical immunosuppressants for multiple years. The subject also tried various topical creams available on the market to no avail. The subject applied daily the topical cream for 4 weeks and exposing the topical cream to light for at least 5 minutes each application. The topical cream used was produced as described in Example 1 but contained 2 wt. % of the plant extract. The eczema and itchiness was completely eliminated afterthe 4 weeks of treatment (Fig. 3A before treatment and Fig. 3B after 4 weeks).

[0073] In another case, the subject presented keratosis and brown spots and was treated with a daily application of the topical cream for 5 weeks with at least 5 minutes of exposure to light each application. The topical cream used was produced as described in Example 1 but contained 1 wt. % of the plant extract. The keratosis was eliminated and the brown spots were significantly reduced after 5 weeks (Figs. 4A-4B).

[0074] In yet another case, the subject presented keratosis and brown spots and was treated with a daily application of the topical cream for 5 weeks with at least 5 minutes of exposure to light each application. The topical cream used was produced as described in Example 1 but contained 1 wt. % of the plant extract. The keratosis was almost completely eliminated and the brown spots were eliminated after 5 weeks (Figs. 5A-5B). Moreover, the arrows in Fig. 5A point to pre-keratosis cells. As can be seen in Fig. 5B, the pre-keratosis cells did not develop into a keratose after treatment with the topical cream and thus the topical cream prevented the onset of keratosis.

[0075] In a further case, the subject presented keratosis and was treated with a daily application of the topical cream for 4 weeks and exposing the topical cream to light for at least 5 minutes each application. The topical cream used was produced as described in Example 1 but contained 1 wt. % of the plant extract. The keratosis was eliminated after 4 weeks leaving only a trace behind (Figs. 6A-6B). [0076] In an additional case, the subject presented redness and lesions and applied the topical cream daily as indicated above for nine days. The lesions were eliminated after nine days and the redness almost disappeared (Figs. 7A-7B).

[0077] Overall the 35 total participants that used the topical cream reported no secondary effects whatsoever.

Claims

WHAT IS CLAIMED IS:

1 . A method of treating, preventing or alleviating the symptoms of a dermatological condition requiring energy supplementation by ATP, the method comprising applying on the skin of a subject in need thereof, a topical cream comprising, as an active ingredient, chloroplasts having functional thylakoid membranes and a gel carrier, and illuminating the topical cream with light.

2. The method of claim 1 , wherein the dermatological condition is selected from redness, itchiness, dryness, irregular skin coloration, milium, firmness, brown spots, wrinkles, rashes, keratosis, inflammation, pigmentation, non-cancerous growths, lesions, eczema and combinations thereof.

3. The method of claim 1 or 2, wherein the topical cream comprises from 0.1 to 10 wt. % of a lyophilised plant extract containing chloroplasts having functional thylakoid membranes, from 67 to 73 wt. % of water, from 4 to 6 wt. % of oils, from 1 to 3 wt. % of essential oils, from 0.5 to 1 .5 wt. % of a gelling agent, from 3 to 5 wt. % of an antimicrobial agent, and from 3 to 7 wt. % of an emulsion agent.

4. The method of claim 3, wherein the topical cream further comprises from 4 to 8 wt. % of an emollient.

5. The method of claim 3 or 4, wherein the topical cream further comprises from 0.1 to 0.3 wt. % of an antioxidant.

6. The method of any one of claims 3 to 5, wherein the topical cream further comprises from 0.5 to 1 .5 wt. % of a cooling agent.

7. The method of any one of claims 3 to 6, wherein the topical cream further comprises from 0.5 to 1 .5 wt. % of a preservative.

8. The method of any one of claims 3 to 7, wherein the topical cream further comprises from 0.25 to 1 wt. % of a ultra-violet (UV) protection agent.

9. A topical cream comprising, as an active ingredient, chloroplasts having functional thylakoid membranes and a gel carrier for use in the treatment, alleviation of symptoms or prevention of dermatological conditions requiring energy supplementation by ATP.

10. The topical cream of claim 9, wherein the dermatological condition is selected from redness, itchiness, dryness, irregular skin coloration, milium, firmness, brown spots, wrinkles, rashes, keratosis, inflammation, pigmentation, non-cancerous growths, lesions, eczema and combinations thereof.

1 1 . The topical cream of claim 9 or 10, wherein the topical cream comprises from 0.25 to 3 wt. % of a lyophilised plant extract containing chloroplasts having functional thylakoid membranes, from 67 to 73 wt. % of water, from 4 to 6 wt. % of oils, from 1 to 3 wt. % of essential oils, from 0.5 to 1 .5 wt. % of a gelling agent, from 3 to 5 wt. % of an antimicrobial agent, and from 3 to 7 wt. % of an emulsion agent.

12. The topical cream of claim 11 , further comprising from 4 to 8 wt. % of an emollient.

13. The topical cream of claim 11 or 12, further comprising from 0.1 to 0.3 wt. % of an antioxidant.

14. The topical cream of any one of claims 1 1 to 13, further comprising 0.5 to 1 .5 wt. % of a cooling agent.

15. The topical cream of any one of claims 11 to 14, further comprising from 0.5 to 1 .5 wt. % of a preservative.

16. The topical cream of any one of claims 11 to 15, further comprising from 0.25 to 1 wt. % of a ultra-violet (UV) protection agent.

17. Use of a topical cream comprising chloroplasts having functional thylakoid membranes and a gel carrier, in the treatment, alleviation of symptoms or prevention of dermatological conditions requiring energy supplementation by ATP.

18. The use of claim 17, wherein the dermatological condition is selected from redness, itchiness, dryness, irregular skin coloration, milium, firmness, brown spots, wrinkles, rashes, keratosis, inflammation, pigmentation, non-cancerous growths, lesions, eczema and combinations thereof.

19. The use of claim 17 or 18, wherein the topical cream comprises from 0.25 to 3 wt. % of a lyophilised plant extract containing chloroplasts having functional thylakoid membranes, from 67 to 73 wt. % of water, from 4 to 6 wt. % of oils, from 1 to 3 wt. % of essential oils, from 0.5 to 1 .5 wt. % of a gelling agent, from 3 to 5 wt. % of an antimicrobial agent, and from 3 to 7 wt. % of an emulsion agent.

20. The use of claim 19, wherein the topical cream further comprises from 4 to 8 wt. % of an emollient.

21 . The use of claim 19 or 20, wherein the topical cream further comprises from 0.1 to 0.3 wt. % of an antioxidant.

22. The use of any one of claims 19 to 21 , wherein the topical cream further comprises from 0.5 to 1 .5 wt. % of a cooling agent.

23. The use of any one of claims 19 to 22, wherein the topical cream further comprises from 0.5 to 1 .5 wt. % of a preservative.

24. The use of any one of claims 19 to 23, wherein the topical cream further comprises from 0.25 to 1 wt. % of a ultra-violet (UV) protection agent.

25. The use of any one of claims 19 to 24, wherein the topical cream is an anti-pollution cream.