Compositions and methods for treating hair loss using non-naturally occurring prostaglandins

A method for treating hair loss in mammals uses compositions containing prostaglandin F analogs. The compositions can be applied topically to the skin. The compositions can arrest hair loss, reverse hair loss, and promote hair growth.

EXAMPLES These examples are intended to illustrate the invention to those skilled in the art and should not be interpreted as limiting the scope of the invention set forth in the claims. 
 Reference Example 1 
 Radioligand Binding Assay IC 50 of a PGF can be determined relative to PGF 2&agr; using the Radioligand Binding Assay. As a control, the IC 50 for PGF 2&agr; itself should be no lower than 1.0 nM and no higher than 5.0 nM. In this assay, COS-7 cells are transiently transfected with the hFP recombinant plasmid using LipofectAMINE Reagent. Forty-eight hours later, the tranfected cells are washed with Hank's Balanced Salt Solution (HBSS, without CaCl 2 , MgCl 2 , MgSO 4 , or phenol red). The cells are detached with versene, and HBSS is added. The mixture is centrifuged at 200 g for 10 minutes, at 4° C. to pellet the cells. The pellet is resuspended in Phosphate-Buffered Saline-EDTA buffer (PBS; 1 mM EDTA; pH 7.4; 4° C.). The cells are disrupted by nitrogen cavitation (Parr model 4639), at 800 psi, for 15 minutes at 4° C. The mixture is centrifuged at 1000 g for 10 minutes at 4° C. The supernatant is centrifuged at 100,000 g for 60 minutes at 4° C. The pellet is resuspended to 1 mg protein/mL TME buffer (50 mM Tris; 10 mM MgCl 2 ; 1 mM EDTA; pH 6.0; 4° C.) based on protein levels measured using the Pierce BCA Protein Assay kit. The homogenate is mixed for 10 seconds using a Kinematica POLYTRON® (available from KINEMATICA AG, Luzernerstrasse147A CH-6014 Littau, Switzerland). The membrane preparations are then stored at −80° C., until thawed for assay use. The receptor competition binding assays are developed in a 96 well format. Each well contains 100 g of hFP membrane, 5 nM (3 H) FPGF2, and the various competing compounds in a total volume of 200 L. The plates are incubated at 23° C. for 1 hour. The incubation is terminated by rapid filtration using the Packard Filtermate 196 harvester through Packard UNIFILTER® GF/B filters (available from Packard Instrument Co., Inc. of Downers Grove Ill.) pre-wetted with TME buffer. The filter is washed four times with TME buffer. Packard Microscint 20, a high efficiency liquid scintillation cocktail, is added to the filter plate wells and the plates remain at room temperature for three hours prior to counting. The plates are read on a Packard TOPCOUNT® Microplate Scintillation Counter (also available from Packard Instrument Co., Inc.) 
 Reference Example 2 
 Telogen Conversion Assay PGF's are tested for their potential to grow hair using the Telogen Conversion Assay. The Telogen Conversion Assay measures the potential of a PGF to convert mice in the resting stage of the hair growth cycle (“telogen”), to the growth stage of the hair growth cycle (“anagen”). Without intending to be limited by theory, there are three principal phases of the hair growth cycle: anagen, catagen, and telogen. It is believed that there is a longer telogen period in C3H mice (Harlan Sprague Dawley, Inc., Indianapolis, Ind.) from approximately 40 days of age until about 75 days of age, when hair growth is synchronized. It is believed that after 75 days of age, hair growth is no longer synchronized. Wherein about 40 day-old mice with dark fur (brown or black) are used in hair growth experiments, melanogenesis occurs along with hair (fur) growth wherein the topical application of hair growth inducers are evaluated. The Telogen Conversion Assay herein is used to screen PGF's for potential hair growth by measuring melanogenesis. Three groups of 44 day-old C3H mice are used: a vehicle control group, a positive control group, and a test PGF group, wherein the test PGF group is administered a PGF used in the method of this invention. The length of the assay is 24 days with 15 treatment days (wherein the treatment days occur Mondays through Fridays). Day 1 is the first day of treatment. A typical study design is shown in Table 3 below. Typical dosage concentrations are set forth in Table 3, however the skilled artisan will readily understand that such concentrations may be modified. 3 TABLE 3 Assay Parameters Group Animal Concen- Application Length of &num; &num; Compound tration volume Study 1 1-10 Test 0.01% in 400 &mgr;L topical 26 days Compound vehicle** 2 11-20 Positive 0.01% in 400 &mgr;L topical 26 days Control vehicle** (T3)* 3 21-30 Vehicle** N/A 400 &mgr;L topical 26 days *(T3) is 3,5,3′-triiodothyronine. **The vehicle is 60% ethanol, 20% propylene glycol, and 20% dimethyl isosorbide (commercially available from Sigma Chemical Co., St. Louis, MO). The mice are treated topically Monday through Friday on their lower back (base of tail to the lower rib). A pipettor and tip are used to deliver 400 &mgr;L to each mouse's back. The 400 &mgr;L application is applied slowly while moving hair on the mouse to allow the application to reach the skin. While each treatment is being applied to the mouse topically, a visual grade of from 0 to 4 will be given to the skin color in the application area of each animal. As a mouse converts from telogen to anagen, its skin color will become more bluish-black. As indicated in Table 4, the grades 0 to 4 represent the following visual observations as the skin progresses from white to bluish-black. 4 TABLE 4 Evaluation Criteria Visual Observation Grade Whitish Skin Color 0 Skin is light gray (indication of initiation of anagen) 1 Appearance of Blue Spots 2 Blue Spots are aggregating to form one large blue area 3 Skin is dark blue (almost black) with color covering majority of 4 treatment area (indication of mouse in full anagen) 
 Example 1 13,14-dihydro-15-(2-benzathiozolyl) pentanor Prostaglandin F 1 &agr;, having the structure: 105 was tested according to the method Reference Example 1. 13,14-dihydro-15-(2-benzathiozolyl) pentanor Prostaglandin F 1 &agr; grew hair and had IC 50 of 45 nM. 
 Comparative Example 1 Latanoprost, having the structure: 106 was tested according to the method Reference Example 1. Latanoprost was active at 0.01% and 0.1%. Grades representing the average animal score on day 26 are reported in Table 5. 
 However, latanoprost is nonselective. Although latanoprost does not negate the effect of activating the FP receptor, latanoprost also activates the EP 1 receptor, which which results in the side effect of causing pain. Example 2 Fluprostenol Methyl Ester having the structure: 107 was tested according to the method Reference Example 1. Fluprostenol grew hair at 0.01% and 0.1%. Grades representing the average animal score on day 26 are reported in Table 5. 5 TABLE 5 Grades Example PGF 0.01% 0.1% Comparative latanaprost 0.71 2.9 Example 1 Example 2 fluprostenol methyl ester 3.9 2.6 
 Comparative Example 2 A composition containing 0.01% of a T3 compound was prepared and tested according to the method of Reference Example 1. The T3 compound grew hair. 
 Example 3 Compositions for topical administration are made, comprising: 6 Component 3-1 3-2 3-3 PGF (wt %) 0.019 0.027 0.045 IC 50 the PGF (nM) 19 27 45 Ethanol (wt %) 59.988 59.983 59.973 Propylene Glycol (wt %) 19.996 19.995 19.991 Dimethyl Isosorbide (wt %) 19.996 19.995 19.991 The PGFs in the compositions are as follows: 7 Sample PGF 3-1 108 3-2 109 3-3 110 A human male subject suffering from male pattern baldness is treated by a method of this invention. Specifically, for 6 weeks, one of the above compositions is daily administered topically to the subject to induce hair growth. 
 Example 4 A composition for topical administration is made according to the method of Dowton et al., “Influence of Liposomal Composition on Topical Delivery of Encapsulated Cyclosporin A: I. An in vitro Study Using Hairless Mouse Skin”, S.T.P. Pharma Sciences, Vol. 3, pp. 404-407 (1993), using a PGF in lieu of cyclosporin A and using the NOVASOME® 1 (available from Micro-Pak, Inc. of Wilmington, Delaware) for the non-ionic liposomal formulation. A human male subject suffering from male pattern baldness is treated each day with the above composition. Specifically, for 6 weeks, the above composition is administered topically to the subject. 
 Example 5 Shampoos are made, comprising: 8 Ex. 5-1 Ex. 5-2 Ex. 5-3 Ex. 5-4 Component Ammonium Lauryl Sulfate 11.5% 11.5% 9.5% 7.5% Ammonium Laureth Sulfate 4% 3% 2% 2% Cocamide MEA 2% 2% 2% 2% Ethylene Glycol Distearate 2% 2% 2% 2% Cetyl Alcohol 2% 2% 2% 2% Stearyl Alcohol 1.2% 1.2% 1.2% 1.2% Glycerin 1% 1% 1% 1% Polyquaternium 10 0.5% 0.25% — — Polyquaternium 24 — — 0.5% 0.25% Sodium Chloride 0.1% 0.1% 0.1% 0.1% Sucrose Polyesters of 3% 3% — — Cottonate Fatty Acid Sucrose Polyesters of 2% 3% — — Behenate Fatty Acid Polydimethl Siloxane — — 3% 2% Cocaminopropyl Betaine — 1% 3% 3% Lauryl Dimethyl Amine 1.5% 1.5% 1.5% 1.5% Oxide Decyl Polyglucose — — 1% 1% DMDM Hydantoin 0.15% 0.15% 0.15% 0.15% PGF having IC 50 of 19 nM — 0.019% 0.019% — PGF having IC 50 of 45 nM 0.045% — — 0.045% Minoxidil 3% 2% Phenoxyethanol 0.5% 0.5% 0.5% 0.5% Fragrance 0.5% 0.5% 0.5% 0.5% Water q.s. q.s. q.s. q.s. The PGF having IC 50 of 19 nM is the same as that in Example 3-1. The PGF having IC 50 of 45 nM is the same as that in Example 3-3. A human subject suffering from male pattern baldness is treated by a method of this invention. Specifically, for 12 weeks, a shampoo described above is used daily by the subject. 
 Example 6 A mascara composition is prepared. The composition comprises: 9 Component % W/W WATER, DEIONIZED, USP q.s. BLACK 1080 MICRONIZED TYPE 10.000 GLYCERYL MONOSTEARATE (2400 TYPE) 8.500 C18-36 ACID TRIGLYCERIDE 5.500 STEARIC ACID, TRIPLE PRESSED, LIQUID 4.000 ETHYL ALCOHOL SD 40-B, 190 PROOF/SERIAL &num;: 4.000 BEESWAX WHITE, FLAKES 3.250 SHELLAC, NF 3.000 LECITHIN, GRANULAR (TYPE 6450) 2.500 TRIETHANOLAMINE 99% - TANK 2.470 PARAFFIN WAX 2.250 PARAFFIN WAX 118/125 2.250 CARNAUBA WAX, NF 2.000 POTASSIUM CETYL PHOSPHATE 1.000 PHENOXYETHANOL 0.800 OLEIC ACID NF 0.750 DL-PANTHENOL 0.350 PVP/VA COPOLYMER 0.250 METHYLPARABEN, NF 0.200 DIAZOLIDINYL UREA 0.200 SIMETHICONE 0.200 ETHYLPARABEN NF 0.150 PENTAERYTHRITYL HYDROGENATED ROSINATE 0.150 PROPYLPARABEN, NF 0.100 TRISODIUM EDTA 0.100 PGF having IC 50 of 19 nM 0.019 The PGF is the same as that used in Example 3-1. A human female subject applies the composition each day. Specifically, for 6 weeks, the above composition is administered topically to the subject to darken and thicken eyelashes. 
 Example 7 Pharmaceutical compositions in the form of tablets are prepared by conventional methods, such as mixing and direct compaction, formulated as follows: 10 Ingredient Quantity (mg per tablet) PGF 0.5 Microcrystalline Cellulose 100 Sodium Starch Glycollate 30 Magnesium Stearate 3 The PGF is the same as that used in Example 3-3. The above composition is administered orally to a subject once daily for 6 to 12 weeks to promote hair growth. 
 Example 8 Pharmaceutical compositions in liquid form are prepared by conventional methods, formulated as follows: 11 Ingredient Quantity PGF 0.1 mg Phosphate buffered physiological saline 10 ml Methyl Paraben 0.05 ml The PGF is the same as that used in Example 3-3. 1.0 ml of the above composition is administered subcutaneously once daily at the site of hair loss for 6 to 12 weeks to promote hair growth. 
 Example 9 A topical pharmaceutical composition is prepared by conventional methods and formulated as follows: 12 Ingredient Amount (wt %) PGF 0.004 Dextran 70 0.1 Hydroxypropyl methylcellulose 0.3 Sodium Chloride 0.77 Potassium chloride 0.12 Disodium EDTA (Edetate disodium) 0.05 Benzalkonium chloride 0.01 HCL and/or NaOH pH 7.2-7.5 Purified water q.s. to 100% The PGF is the same as that used in Example 3-3. The above composition is administered ocularly to a subject once per day for 6 to 12 weeks to promote eyelash growth. 
 Effects of the Invention The compositions and methods herein provide a cosmetic benefit with respect to hair growth and appearance in subjects desiring such treatment.