Body Sculpting

Abstract

The invention pertains to a pharmaceutical composition for topical administration, comprising a prodrug for an agonist and/or an antagonist for an adrenergic receptor, wherein the prodrug has an octanol/water partition coefficient of at least 0, for use in a method of shaping a mammalian body by modulation of subcutaneous fat tissue. The invention further pertains to cosmetic and therapeutic application of such prodrugs, such as their use in methods of shaping a mammalian body by locally modulating subcutaneous fat tissue. The invention also pertains to the prodrugs themselves, as well as to methods of making these prodrugs.

Claims

1. A pharmaceutical composition for topical administration, comprising a prodrug for an agonist and/or an antagonist for an adrenergic receptor, wherein the prodrug is an ester, which prodrug comprises said agonist or antagonist and a hydrolyzable moiety, wherein the prodrug has an octanol/water partition coefficient of at least 0, for use in a method of shaping a mammalian body by modulation of subcutaneous fat tissue.

2. The pharmaceutical composition according to claim 1, wherein the modulation occurs at the site of topical administration.

3. The pharmaceutical composition according to claim 1, wherein modulation comprises decreasing the quantity of subcutaneous fat tissue, increasing the quantity of subcutaneous fat tissue, or reinforcing subcutaneous fat tissue.

4. The pharmaceutical composition according to claim 3, wherein modulation comprises decreasing the quantity of subcutaneous fat tissue.

5. The pharmaceutical composition according to claim 1, wherein the prodrug has an octanol/water partition coefficient of at least 2.3.

6. The pharmaceutical composition according to claim 1, wherein the agonist is a an agonist for a beta adrenergic receptor (“beta-agonist”) or an agonist for an alpha-adrenergic receptor (“alpha-agonist”), and/or wherein the antagonist is a antagonist for the beta-adrenergic receptor (“beta-antagonist”) or an antagonist for the alpha-adrenergic receptor (“alpha-antagonist”).

7. The pharmaceutical composition according to claim 6, wherein the beta-agonist is octopamine (ortho-, meta- or para-octopamine, preferably para-octopamine), synephrine (ortho-, meta- or para-synefrine, preferably para-synephrine), norepinephrine, epinephrine, ephedrine, phenylpropanolamine, tyramine, epinine, phenylethanolamine, beta-phenylethylamine, hordenine, isopropylnorsynephrine, N-methyltyramine, salbutamol, levosalbutamol, terbutaline, pirbuterol, procaterol, clenbuterol, metaproterenol, fenoterol, bitolterol, ritodrine, isoprenaline, salmeterol, formoterol, bambuterol, clenbuterol, olodaterol, indacaterol, Amibegron (SR-58611A), CL 316,243, L-742,791, L-796,568, LY-368,842, Mirabegron (YM-178), Ro40-2148, CGP12177, Solabegron (GW-427,353), BRL 37,344; the alpha antagonist is Aripiprazole, Asenapine, Atipamezole, Cirazoline, Clozapine, Efaroxan, Idazoxan, Lurasidone, Melperone, Mianserin, Mirtazapine, Napitane, Olanzapine, Paliperidone, Risperidone, Phenoxybenzamine, Phentolamine, Piribedil, Rauwolscine, Risperidone, Rotigotine, Quetiapine, Norquetiapine, Setiptiline, Tolazoline, Yohimbine, Ziprasidone or Zotepine; the beta-antagonist is Carteolol, Nadolol, Penbutolol, Pindolol, Propranolol, Sotalol, Timolol, Acebutolol, Atenolol, Betaxolol, Bisoprolol, Celiprolol, Esmolol, Metoprolol, Nebivolol, Bucindolol, Carvedilol, Labetolol, preferably Penbutolol, Pindolol, Propranolol, Atenolol, Metoprolol L-748,328, L-748,337, SR 59230A; the alpha-agonist is 4-NEMD, 7-Me-marsanidine, Agmatine, Apraclonidine, Brimonidine, Clonidine, Detomidine, Dexmedetomidine, Fadolmidine, Guanabenz, Guanfacine, Lofexidine, Marsanidine, Medetomidine, Methamphetamine, Mivazerol, Rilmenidine, Romifidine, Talipexole, Tizanidine, Tolonidine, Xylazine, Xylometazoline, TDIQ.

8. (canceled)

9. The pharmaceutical composition according to claim 1, wherein the ester is a C2-C32 alkyl ester.

10. The pharmaceutical composition according to claim 1, wherein the ester is a butanoate, pentanoate, heptanoate, octanoate or decanoate ester.

11. The pharmaceutical composition according to claim 1, wherein the prodrug is present in the composition at a concentration of 0.001-1000 mg/ml.

12. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a cream, foam, gel, lotion, ointment, patch, paste, solution or spray.

13. The pharmaceutical composition according to claim 1, wherein the prodrug is a beta-agonist.

14. The pharmaceutical composition according to claim 13, wherein the beta-agonist is octopamine or synefrine, preferably p-octopamine or p-synefrine.

15. The pharmaceutical composition according to claim 1, further comprising a phosphodiesterase inhibitor and/or an adenyl cyclase stimulator.

16. A method of shaping a mammalian body by locally modulating subcutaneous fat tissue, comprising topically administering a pharmaceutical composition as defined in claim 1.

17. The method according to claim 16, wherein the method is a cosmetic method.

18. The method according to claim 16, wherein the prodrug is administered at a dosage of 0.001-1000 mg/cm2.

19. A prodrug for octopamine, wherein the prodrug is an ester comprising octopamine and a hydrolyzable moiety, wherein the prodrug has an octanol/water partition coefficient of at least 0.

20. (canceled)

21. The prodrug according to claim 19, wherein the prodrug has an octanol/water partition coefficient of at least 2.3.

22.-24. (canceled)

25. The prodrug according to claim 19 for medical use.

26. The prodrug according to claim 19 for use in a method of shaping a mammalian body by modulation of subcutaneous fat tissue.

27. A method for decreasing the quantity of subcutaneous fat tissue, increasing the quantity of subcutaneous fat tissue, or reinforcing subcutaneous fat tissue comprising administering the prodrug of claim 19 to a mammal.

28. A method of making a prodrug for an octopamine, comprising esterifying octopamine with an acylating agent.

Description

EXAMPLE 1: POLARITY CALCULATIONS OF ESTER PRODRUGS

[0152] Polarity calculations were made of different esters of the parent compound. Table 1 depicts the results of these calculations. Lipophilicity increased with butanoate, pentanoate, heptanoate and decanoate by generally a log P of 1, 1.5, 2.5 and 4 respectively.

TABLE-US-00001 TABLE 1 LogP calculations of polarity of ester prodrugs (ACD Chemsketch calculated logP). Octopamine Synephrine Isoprenaline Freebase −0.28 −0.03 0.25 Butanoate 0.86 1.11 1.04 Pentanoate 1.39 1.64 1.57 Heptanoate 2.45 2.70 2.63 Decanoate 4.05 4.29 4.23

EXAMPLE 2: IN VITRO ESTER HYDROLYSIS ASSAY

[0153] Abdominal fat tissue was harvested from male wistar rats (400 g, Harlan Zeist) and stored at −80° C. until use. Human fat tissue was obtained from esthetic surgery and stored at −80° C. until use. Tissue was thuraxed (80 mg/ml for rat tissue and 240 mg/ml for human tissue) for 1 min in Phosphate buffered saline (PBS (Irvine Scientific).

[0154] Octopamine released was measured using a 50 ml beaker thermostated at 30 degrees. 30 ml PBS (stirred) was pumped at 1 ml/min (Gilson minipulse 3) through a UV detector (SPD-10Avp Shimadzu UV/VIS) set at 280 nm and 2.56 AUFS. The outlet was recirculated back to the beaker. UV signal was recorder on a flatbed recorder (Kipp), set at 1 mm/min and 1 mV gain.

[0155] Octopamine and octopamine decanoate solutions (1 mM) were made in ultrapure water adding 1 microliter/ml of 1.8% hydrochloride acid. Octopamine decanoate was sonicated for 30 min at 30 degrees.

[0156] Upon stabilization of the UV detector on PBS, 1 ml of tissue suspension was added to the PBS. Upon stabilization of the UV signal, 5 ml of 1 mM of octopamine decanoate was added. Control experiments were performed without addition of tissue suspension in order to monitor spontaneous hydrolysis of decanoate ester in PBS.

Results

[0157] UV spectra were taken from octopamine and octopamine decanoate. Octopamine showed a UV absorption maximum at 279 nm, whereas octopamine decanoate showed a maximum at 269 nm. Relative intensity of octopamine was about 10 times higher than octopamine decanoate.

[0158] FIG. 1 shows the results of the in vitro hydrolysis experiments. Octopamine decanoate spontaneously hydrolyzed into octopamine at a rate of 10% per 110 min. During presence of rat fat suspension, octopamine decanoate hydrolyzed much faster, yielding over 50% of free octopamine within 110 min. In presence of human fat tissue at concentrations three times higher than rat tissue, decanoate hydrolyzed yielding over 40% conversion in 110 min.

[0159] From this, it is apparent that hydrolysis of an ester prodrug of octopamine, octopamine decanoate, is faster in the presence of fat tissue than in PBS. This must be caused by the presence of fat tissue, such as for instance endogenous enzymes, among which lipase, which is known to hydrolyze esters.

EXAMPLE 3 IN VITRO ASSAY OF EFFECT OF OCTOPAMINE DECANOATE ON GLYCEROL PRODUCTION IN HUMAN FAT

[0160] Human fat tissue was pottered using a teflon potter in 10 ml PBS with 6 mM Glucose (2.4 gram fat tissue per 10 ml). The suspension was transferred to a 50 ml beaker and was gently stirred at 37 degrees Celsius to maintain the homogeneous nature. 0.3 ml suspension was transferred to 2 ml reaction vials, spiked with octopamine (end concentration 1 microM) and octopamine decanoate (end concentration 10 microM and PBS/glucose were added until 2 ml end volume. Vials were incubated at 37 degrees Celsius for 2 hours.

[0161] Samples were spun off at 4000 rpm for 2 min and 10 microliter samples were taken after removal of floating fat with a tissue.

[0162] Samples were analyzed using an enzymatic kit (Sigma Aldrich) and fluorescence intensity was measured using direct flow injection (Vici valve in combination with Shimadzu 10 ADvp HPLC pump at 0.15 ml/min, thermo 15*2.1 C18 column prior to valve and using ultrapure water as mobile phase) of 20 microliter into a HPLC fluorescence detector (Shimadzu RF 10Axl). Calibration was performed by preparation of calibration samples 2-1000 microM.

Results

[0163] FIG. 2 shows the effect of octopamine and octopamine decanoate on glycerol production in human fat suspension. Levels increased after 2 hours of incubation with octopamine (P=0.10, two tailed t-test) and reached significance for octopamine decanoate (P=0.047, two tailed t-test).

EXAMPLE 4 IN VITRO ASSAY OF PENETRATION OF OCTOPAMINE DECANOATE THROUGH HUMAN SKIN

[0164] 4 by 4 cm square of human skin were cut and washed with PBS. Subcutaneous fat was removed and the skin was fixated over a 50 ml stirred beaker containing PBS filled so there was no air between PBS and the inside of the skin. The open surface of the skin that was exposed to the outside was 4.9 cm.sup.2. To the PBS 1 ml of 0.221 mg/ml human fat that was thuraxed in PBS was added.

[0165] After stabilization of 0.5 hrs, 1 ml of hydrogel comprising 5 mg/ml octopamine decanoate was administered by gently rubbing in the skin for 1 min. The skin was covered by an inverted 20 ml beaker and left overnight for penetration and hydrolysis to occur. Samples were taken at t=0 and t=1000 min and immediately frozen at −80 degrees Celsius.

[0166] Samples were analyzed using HPLC with UV detection. A Shimadzu HPLC pump (10 ADvp) was used in combination with a valco injection valve (20 microliter loop) with a Thermo HPLC column (150 mm*2.1 mm, BDS hypersil, C18). Octopamine was detected at 279 nm and calibration occurred by injection of standards 0-1000 microM. The mobile phase consisted of 786 mg KH.sub.2PO.sub.4, 500 ml ultrapure water, 15 ml MeOH, 0.5 ml acetic acid (99%) and 55.55 mg heptasulfonic acid and pumped through the system at 0.25 ml/min.

Results

[0167] Concentrations of octopamine that were detected in the beaker 1000 min after application of the octopamine decanoate hydrogel were 0.524 microM (0.294 sem). Percentual penetration/conversion was calculated to be 0.18+0.09%.

[0168] From this, it follows that the octopamine decanoate prodrug penetrates the skin and is hydrolyzed after penetration by the presence of fat tissue, among which lipase.

EXAMPLE 5: IN VIVO EFFECT OF OCTOPAMINE DECANOATE HYDROGEL TREATMENT ON WAIST AND WEIGHT OF RATS

[0169] Male wistar rats (approx. 500 g) were weight daily and waistline was measured. Animals were shaven once weekly at least 12 hours before next treatment to ensure wound healing.

[0170] Animals were first treated for 1 month with carbomer hydrogel not containing prodrugs (twice daily abdominal application 3 by 3 cm), after which animals were treated with 0.5 ml carbomer hydrogel containing 5 mg/ml octopamine decanoate (application concentration 0.277 mg/cm.sup.2). Tail vena blood draws (100 microliter plasma, 5 microliter heparine 500 IE per 100 microliter blood) were taken on the day −8, 21, 36 (start of compound treatment), 51 and 58. Blood was spun off (10 min 14 KRPM) and plasma was stored at −80.

[0171] After 3 weeks of treatment with octopamine decanoate (16 hrs after last application), animals were anaesthetized using isoflurane (2%, 0.81/min 02) and microdialysis probes (2 cm cellulose membrane, Brainlink, the Netherlands) were inserted in abdominal fat for measurement of octopamine. Probes were perfused with saline at 1.5 microliter per min and 30 min samples were collected in 300 microliter vials. Sample collection was commenced 15 min after insertion of the probe.

Analysis

[0172] Blood and dialysate samples were analyzed for octopamine using LC-MSMS (Shimadzu 20 ADvp in conjunction with Sciex API 4000) after derivatization with SymDAQ. Briefly, 22.5 microliter samples were mixed with 0 microliter 0.5 mg/ml SymDAQ reagent and injected onto the column. Calibration was performed with samples from 0.01-8 nM.

[0173] Blood samples were analyzed for glycerol using an enzymatic kit (Sigma Aldrich) and fluorescence intensity was measured using direct flow injection (Vici valve in combination with shimadzu 10 ADvp hplc pump at 0.15 ml/min, thermo 15*2.1 C18 column prior to valve and using ultrapure water as mobile phase) of 20 microliter into a HPLC fluorescence detector (Shimadzu RF 10Axl). Calibration was performed by preparation of calibration samples 2-1000 microM.

Results

[0174] FIG. 3 shows the effect of treatment of animals with control carbomer hydrogel followed by 5 mg/ml octopamine decanoate hydrogel. While animal weights remained inclining according to their growth curve, waistlines significantly reduced from initiation of application of octopamine carbomer hydrogel, reaching a reduction of about 10% after 3 weeks of treatment. Waistline was significantly reduced when compared to control treatment using a one way anova P<0.001 (1-way ANOVA RM-post hoc Bonferroni; p=0.05).

[0175] FIG. 4 shows the effect of treatment with octopamine decanoate carbomer hydrogel on plasma glycerol levels. Levels increased after 15 days (P=0.101, t-test two tailed), reaching significance after 22 days of treatment (P=0.024, t-test, two tailed).

[0176] Plasma levels of octopamine were under LLOQ (lower limit of quantification (0.1 nM) both before initiation of treatment with octopamine decanoate carbomer hydrogel, as after 15 and 22 days of treatment. This illustrates that treatment does not lead to significant systemic octopamine exposure. Subcutaneous levels of octopamine were 0.96 nM±0.36 nM, exceeding plasma levels even 16 hours after the last application.

[0177] It follows that topical administration of an octopamine prodrug according to the invention decreases the quantity of subcutaneous fat tissue, while not leading to increased systemic concentrations of free octopamine.

EXAMPLE 6: HYDROLYSIS EXPERIMENTS (FIGS. 6, 7, 8, 9 AND 10)

[0178] Human belly adipose tissue was obtained from obese female subjects that underwent esthetic surgery. Tissue was pottered (Potter RW 19 Nr 29795 of Janke & Kunkel KG) in PBS solution (Irvine Scientific) with 5 mM D(+)-glucose monohydrate at 530 rpm using a Teflon potter tip.

[0179] Tissue suspension (1, 7.5, 25 or 240 mg fat/ml PBS, or plasma) was stirred and 1.98 ml aliquots were transferred to polypropylene 2 ml screwcap vials (Sarstedt). Prodrug esters (octopamine decanoate and synephrine decanoate) were added at the indicated concentration, mixed and incubated at 37° C. (FIG. 6). When inhibition of lipase was studied, propranolol or orlistat were added 5 minutes before addition of the prodrug esters (FIG. 10).

[0180] 15 minutes after addition of the prodrug, lipolysis was stopped by adding 20 microliter of 1.8% HCl. Samples were spun down (13000 rpm for 3 min at 4° C.) and supernatants were stored at −18° C. for analysis. For analysis of hydrolysis in plasma (FIG. 7), 10 microliter whole blood was added to 1 ml of PBS comprising octopamine decanoate at the indicated concentration. For stability of esters, octopamine and synephrine in PBS (FIGS. 8 and 9), incubation was performed in PBS without fat or blood.

[0181] Samples were analyzed using HPLC UV. A Gilson 234 auto-injector and Shimadzu hplc pump (10 AdVP) was used in conjunction with a shimadzu UV (10 AVp) set at 270 nm. Samples were separated using a reversed phase HPLC column (Thermo BDS Hypersil C18 150 mm×2.1 mm, 3 micrometer). The mobile phase consisted of 1.6 g KH.sub.2PO.sub.4, 110 mg sodium 1-heptane sulfonate, 1 l ultrapure water, 15 mL methanol and 1 ml acetic acid at a flowrate of 0.175 ml/min.

Results

[0182] Increasing quantities of fat tissue in suspensions results in a higher hydrolysis rate of octopamine decanoate and synephrine decanoate (FIG. 6 a-d). It follows that both octopamine decanoate and synephrine decanoate are hydrolyzed by the presence of fat tissue, in particular by endogenous enzymes present in fat tissue, in particular lipase.

[0183] Octopamine decanoate can also be hydrolyzed in plasma (FIG. 7), which ensures that the little amount of prodrug that enters the bloodstream is rapidly converted to free octopamine, so prodrugs do not distribute throughout the body.

[0184] Hydrolysis of octopamine decanoate (“octdec”), octopamine pentanoate (“octpent”) and synephrine decanoate (“syndec”) occurs in PBS at a much lower rate than in the presence of plasma or fat tissue (FIG. 8). This indicates that endogenous compounds, most likely enzymes such as lipase, are responsible for the increased hydrolysis of prodrugs of the invention into active adrenergic receptor agonists and/or antagonists.

[0185] Free octopamine or synephrine is stable in PBS (FIG. 9).

[0186] The self-reinforcing, auto-catalytic effect of administration of prodrugs according to the invention can be shown as follows. If octopamine decanoate (“OD”) or synephrine decanoate (“SD”) is added to a fat suspension as described above, a base level of about 40-45% hydrolysis is observed after 15 minutes (FIG. 10).

[0187] Propranolol is a beta receptor antagonist. The beta antagonistic action of propranolol has the effect of suppressing lipase action by receptor mediation. It has been shown that addition of increasing quantities of propranolol decreases the hydrolysis of octopamine decanoate (FIG. 10a) or synephrine decanoate (FIG. 10c), relative to the control. Consequently, lipase from fat tissue is at least partially responsible for the hydrolysis of octopamine decanoate and synephrine decanoate in the presence of fat tissue.

[0188] This is even more apparent when adding orlistat to the suspension. Orlistat is a lipase antagonist, and consequently blocks lipase itself. It has no effect on the beta receptor-mediated stimulation or suppression of lipase. Addition of Orlistat to a suspension of octopamine decanoate (FIG. 10b) or synephrine decanoate (FIG. 10d) further suppresses the hydrolytic action of lipase on octopamine decanoate or synephrine decanoate. By blocking lipase itself, hydrolysis of the prodrug is suppressed to a greater extent than by blocking the beta receptor, which only has the effect of depressing lipase activation.

[0189] These results should be evaluated in context. The hydrolysis product, octopamine or synephrine, has itself the effect of stimulating the beta adrenergic receptor thereby stimulating lipase activity, as can be seen by for instance the increase in glycerol production (FIGS. 2 and 4). Lipase activity is also responsible for the hydrolysis of the prodrugs of the invention (FIGS. 6 a-d).

[0190] It follows that topical administration of prodrugs of the invention results in local hydrolysis of the prodrug to result in free octopamine or synephrine, which results in increased lipase activity. Increased lipase activity is responsible for increased hydrolysis of the prodrug, as well as increased hydrolysis of triglycerides. Thus, the prodrug of the invention is autocatalytic in driving its own hydrolysis by activation of lipase, and this activation concomitantly results in an increased hydrolysis of triglycerides (fat tissue). Thus, the action of the prodrug on lipase stimulates the lipase action on the prodrug, resulting in much increased hydrolysis of triglycerides. There is, therefore, a distinct synergy between lipase activation and hydrolysis of the prodrug.

[0191] In addition, due to the high log P of the prodrugs of the invention, the prodrugs are absorbed preferentially in fat tissue, where lipase is to be found and where triglycerides are to be hydrolyzed. This results in highly efficient hydrolysis of triglycerides, in accordance with the present claims. Thus, there is a further synergy between the autocatalytic hydrolysis mechanism described above, and the prodrug property log P, which is responsible for the partitioning of the prodrug in fat tissue.

[0192] It is postulated that it is reasonable to expect that the opposite effect, increasing the quantity of subcutaneous fat tissue by depressing lipase activity, may also occur. This is because it follows from the described experiments that suppressing lipase activity is never 100%, so that some remnant lipase activity remains. Thus, also in case of agonists or antagonists that suppress lipase activity (beta-antagonists and/or alpha agonists as described above), some lipase activity remains, allowing for hydrolysis of the prodrug and further depressing lipase activity. This would result in locally increasing the quantity of subcutaneous fat tissue, and/or reinforcing subcutaneous fat tissue.

[0193] However, as the autocatalytic effect is strongest for agonists and a antagonists that stimulate lipase activity (beta agonists and/or alpha antagonists as described above), prodrugs that stimulate lipase activity are preferred.

EXAMPLE 7: SKIN PENETRATION

[0194] Human skin from obese female subjects that underwent esthetic surgery was dissected and clamped in a skin penetration chamber which allowed 6.25 cm.sup.2 skin exposed over 60 ml stirred PBS (5 mM glucose) which contained 7.5 mg/ml pottered tissue for conversion of prodrugs upon penetration. 0.25 ml of a hydrogel comprising 2.75 mg/ml free octopamine, 5 mg/ml octopamine decanoate or 3.98 mg/ml octopamine pentanoate was applied to the skin once and left to penetrate for 2 hours, ensuring full hydrolysis of the prodrugs to free octopamine. The chamber was thermostated at 37° C. Samples were drawn from the PBS pottered tissue with a 1 ml syringe and analyzed using LC-mass spectrometry. Given the full conversion, this assay evaluates the total penetration of octopamine prodrug through skin based on equal amounts of octopamine, and compares this to the penetration of an equal amount of octopamine itself.

[0195] Samples were analyzed using HPLC masspectrometry. A Shimadzu HPLC (LC20AD pump and SIL 10 ADvp injector) was used in conjunction with a sciex API 4000 Masspectrometer. The HPLC column was a Phenomenex, Synergi Max (BOL-P-RP2.5-036), 3.0×100 mm, 2.5 m, thermostated at 35° C. The mobile phase consisted of Eluent A: 0.1% formic acid (“FA”) in ultrapure water (“UP”) and Eluent B: 70% acetonitrile (“ACN”)+0.1% FA at a total flow of 0.3 ml/min. The make-up flow consisted of Eluent C: 0.1% FA in ACN at 0.15 ml/min and Rinsing liquid: UP/ACN/FA=50/50/0.1.

[0196] Separation of octopamine was accomplished by running the gradient from 0 to 40% eluent B in 4 min and than to 100% eluent B in the next 1.5 min. Gradients were maintained at 100% B for 0.5 minute.

[0197] Octopamine was determined after precipitation (25 nM octopamine-d3 in ACN/UP/FA 95%/5%/0.1%. 10 μL sample was added to 15 μL precipitation solvent and vortexed for 10 sec. Samples were centrifugated for 5 mins at 13000 rpm and 14 μL 0.1% FA in UP was added to 6 μL supernatant and vortexed for 10 sec. The autoinjector was programmed to add 20 μl 0.5 mg/ml SymDAQ reagent (online) and inject 35 μl. The SymDAQ reagent was prepared by dissolving 5 mg SymDAQ in 4.5 ml UP, 5 ml 0.25 M NaHCO.sub.3, 0.5 ml methanol (“MeOH”) and 20 microliter 2-mercapto-ethanol.

TABLE-US-00002 TABLE 1 Settings of MSMS Analyte Q1 Q3 Dwell time (ms) DP EP CE CXP Octopamine 399 356 100 91 10 33 24 fragment 356 Octopamine 399 278 50 91 10 49 20 fragment 278 Octopamine-d3 402 359 100 91 10 33 15

TABLE-US-00003 TABLE 2 Settings of MSMS Probe position x = 4, y = 1.5 Curtain gas 20 (N.sub.2) CAD gas (N.sub.2) 8 GS1 (nebulizer, 40 zero air) GS2 (zero air) 15 IS voltage 5500 ihe On Temperature 600 Resolution Q1 Unit Resolution Q3 Unit MR pause 5 ms Settling time 2 ms

Results

[0198] Topical administration of a hydrogel comprising free octopamine resulted in minor penetration of octopamine through skin. Penetration of octopamine decanoate and pentanoate was about a factor 10 higher (FIG. 11).

EXAMPLE 8: SKIN AND FAT PENETRATION ASSAY

[0199] Cubes of human fat (5×5×5 cm) with skin attached from obese female subjects that underwent esthetic surgery was dissected at 4° C. Cubes were transferred to containers so the skin would overlay the rim of the container. 0.25 ml of hydrogels comprising 2.75 mg/ml free octopamine, 5 mg/ml octopamine decanoate or 3.98 mg/ml octopamine pentanoate were applied once and left to penetrate for 20 hrs. Cubes were subsequently frozen at −80° C.

[0200] Upon defrosting, skin was carefully removed taking care not to contaminate the underlying fat. The fat was dissected to yield a column of 1×1 cm of fat that was directly under the site of application. The column was sliced to yield 0.5 cm thick slices covering 0-2 cm fat depth under the site of application. Tissue was sonicated in 5 ml PBS (5 mM glucose), samples were spun down (13000 rpm for 30 min at 4° C.). Clear supernatant was removed with an injection needle and syringe and frozen until analysis. Analysis of octopamine was performed as described in Example 7.

Results

[0201] Octopamine, octopamine decanoate and octopamine pentanoate penetrate through both skin and fat tissue. Application of octopamine decanoate and pentanoate results in higher concentrations of free octopamine in all fat layers than application of a hydrogel comprising octopamine (FIG. 12).

EXAMPLE 9: IN VITRO GLYCEROL PRODUCTION ASSAY

[0202] Human belly adipose tissue obtained from obese female subjects that underwent esthetic surgery was pottered (Potter RW 19 Nr 29795 of Janke & Kunkel KG) in PBS solution (Irvine Scientific) with 5 mM D(+)-glucose monohydrate at 530 rpm using a Teflon potter tip, to give a 250 mg/ml human fat suspension.

[0203] The fat suspension was stirred and 1.75 ml aliquots were transferred to polypropylene 2 ml screwcap vials (Sarstedt). Test compounds were added at a concentration as indicated and vials were incubated for 4 hrs at 37° C. Vials were mixed every hour. Glycerol production in 4 hrs was calculated by analyzing glycerol content of the control (a suspension which was not incubated but instead immediately frozen) vs control samples that were incubated for 4 hours. The produced glycerol quantity was set as 100%. The glycerol content of experimental samples was expressed as % of the control.

[0204] Experimental samples were frozen after incubation. The fat pellet was removed and upon defrosting, samples were spun down (13000 rpm for 15 min at 4° C.), and clear supernatant was pipetted off and frozen until analysis.

[0205] Glycerol was analyzed using an enzymatic kit (Glycerol Assay Kit, Sigma Aldrich). Briefly, in a 96 well plate (Corning), 100 microliter glycerol assay reaction mix was added to 10 microliter samples of supernatant and left to incubate for 20 minutes after shaking for 15 seconds. Absorbance was read by a platereader (Thermo Multiskan FC) at 570 nm. A glycerol calibration line (0.015 microM-1000 microM) was used for quantification.

Results

[0206] Isoprenaline resulted in an increase in glycerol content at concentrations varying from 0.1 to about 800 nM (FIG. 13a).

[0207] Octopamine resulted in an increase in glycerol content at concentrations varying from 0.1 to 100000 nM (FIG. 13b).

[0208] Synephrine resulted in an increase in glycerol content at concentrations varying from 0.1 to at least 100000 nM (FIG. 13c).

[0209] Propranolol resulted in a more or less constant glycerol content at concentrations from 0.1 to at least 1000 nM (FIG. 13c). At concentrations above 1000 nM, glycerol content decreased.

[0210] Beta 3 agonists (SR 58611A, CL 316243, CGP12177) increased glycerol production whereas beta antagonist SR 59230 reduced glycerol production (FIG. 20).

[0211] Alpha 2 antagonist yohimbine increased glycerol production whereas alpha 2 agonist xylazine reduced glycerol production (FIG. 20).

[0212] Phosphodiesterase inhibitor caffeine increased glycerol production (FIG. 20).

[0213] From these results, it can be seen that addition of beta adrenergic receptor agonists octopamine, synephrine and isoprenaline results in hydrolysis of triglycerides to give an increased content of glycerol. This effect increases with increasing concentration of agonist, but decreases at very high concentration. It is presently assumed that desensitization of the beta receptor is responsible for the decrease in glycerol content at high concentrations of beta adrenergic receptor agonists.

[0214] Addition of the beta adrenergic receptor antagonist propranolol has the opposite effect: there is a decrease in glycerol content at concentrations above 1000 nM, and this effect increases with increasing concentration. It is assumed that antagonists with higher antagonistic activity than propranolol will display this effect at lower concentrations.

[0215] From FIG. 20, it can be seen that the effects reported here also occur for other beta agonists, as well as for alpha antagonists. The opposite effect occurs for beta antagonists and alpha agonists, as has been described above.

EXAMPLE 10: APPLICATION ON HUMAN VOLUNTEERS

[0216] For abdominal/hip experiments 2 male volunteers 43 and 39 years old (BMI 25.5 and 28 resp.) monitored belly circumference, and skinfold at belly and hip on a daily basis (8 am mornings). Blood pressure and body weight was monitored daily. For leg experiments 1 female volunteer (42) years old (BMI 23.1) monitored leg circumference on a daily basis (8 am mornings).

Application of Hydrogel

[0217] Hydrogel was applied daily or twice daily as indicated using a 5 ml syringe to measure volume.

[0218] For abdominal experiments, the indicated volume of hydrogel was applied on the belly around the belly button in a radius of 10 cm. The same volume was applied for hip areas. For leg experiments, 2.5 ml was applied on each leg.

Plasma Sampling

[0219] After thorough washing of hands, blood was sampled using fingerprick. Blood (40 microliter) was sampled using a capillary that protruded the cap of the vial and spun down into 300 microliter vials containing 10 microliter of heparin 20 IE/ml. Plasma was pipetted off and transferred to 300 microliter vials and stored at −20° C. until analysis.

[0220] Octopamine was analyzed as described in Example 7.

Plasma Glycerol Analysis

[0221] Glycerol was analyzed using a enzymatic kit (Sigma), as described above.

Skinfold

[0222] Skinfold was assessed by measuring thickness of skin at hips and belly 4 cm from belly button using a skinfold measuring device (Vetmeter Slimguide C-120).

Treatment Regime

[0223] The effect of application on belly and hips of a hydrogel comprising 2.75 mg/ml free octopamine (2.5 ml, once daily), of a hydrogel comprising 5 mg/ml octopamine decanoate, (2.5 ml, once daily and 5 ml, twice daily) on waistline of humans was studied (FIG. 14). Experiments are the average of two experiments (n=2).

[0224] Also, the effect of application on belly and hips of control hydrogel, 2.75 mg/ml octopamine (2.5 ml, once daily), of 5 mg/ml octopamine decanoate (2.5 ml, once daily and 5 ml, twice daily) and 3.98 mg/ml octopamine pentanoate (2.5 ml, once daily) and of 5 mg/ml synephrine decanoate (2.5 ml, once daily) on waistline of a single individual was studied (FIG. 15).

[0225] Also, the effect of 5 mg/ml octopamine decanoate (5 ml, twice daily) on belly and hip skinfold was studied (FIG. 16). Experiments are an average of 2 or 3 runs (n=2-3).

[0226] Also, plasma levels of octopamine were monitored upon administration of octopamine decanoate (5 mg/ml, 2.5 ml once daily and 5 ml, twice daily; FIG. 17a); of free octopamine (2.75 mg/ml, 2.5 ml, once daily and 5 ml, twice daily; FIG. 17b), and of octopamine pentanoate (3.98 mg/ml, 2.5 ml, once daily); FIG. 17c). Experiments are an average of one or two experiments (n=1-2).

[0227] Experiments were set up to compare equal amounts of free octopamine. Thus, the quantity of prodrug or octopamine is varied so as to provide equal amounts of free octopamine.

Results

[0228] Topical administration of hydrogels comprising octopamine decanoate (“octdec”) decreased the waistline by about 4% after 16 days, irrespective of whether 2.5 ml once daily or 5 ml twice daily was used. Topical administration of free octopamine (“oct”) in the same treatment regime and at the same molar concentration was without effect (FIG. 14).

[0229] Topical administration of hydrogels comprising octopamine decanoate (“octdec”), octopamine pentanoate (“octpent” or of synephrine decanoate (“sydec”) decreased the waistline by 2-5% after 21 days. Topical administration of free octopamine (“oct”) in the same treatment regime and at the same molar concentration was without effect (FIG. 15).

[0230] Topical administration of hydrogels comprising octopamine decanoate reduced skinfold by 10-20% in 20 days (FIG. 16).

[0231] The treatment regimes using hydrogels comprising octopamine decanoate and pentanoate prodrugs did not result in unacceptable plasma levels of free octopamine (FIGS. 17a and c). Octopamine was slowly released from fat tissue to plasma, and no side effects were reported. The administration of prodrugs resulted in acceptable systemic concentrations of free octopamine, which was also the case for administration of a hydrogel comprising free octopamine (FIG. 17b). However, the hydrogel comprising free octopamine dis not result in a decrease of subcutaneous fat tissue (FIG. 15).

[0232] During chronic treatment with a hydrogel comprising octopamine decanoate (5 mg/ml, 5 ml, twice daily), plasma levels of octopamine remained at acceptable values. No side effects were reported (FIG. 18).

[0233] Blood pressure (systole (“syst”) and diastole (“dia”)) and heart rate (“HR”) remained normal during these experiments (FIG. 19), and no side effects were reported.

EXAMPLE 11: REMOVAL OF CELLULITE

[0234] A hydrogel comprising 3.98 mg/ml octopamine pentanoate (5 ml) was applied once daily on a human subject in an area with moderate cellulite. After three days the cellulite was noticeably decreased.

FIGURES

[0235] FIG. 1: hydrolysis of octopamine decanoate (“octdec”) in phosphate buffered saline (“PBS”), PBS/rat fat suspension or PBS/human fat suspensions (diamond n=3, square n=2, triangles n=2).

[0236] FIG. 2: effect of incubation of PBS/human fat suspension with octopamine or octopamine decanoate on glycerol production

[0237] FIG. 3: effect of treatment of rats with carbomer hydrogel (0.5 ml on 3×3 cm) followed by 5 mg/ml octopamine decanoate in carbomer hydrogel (0.5 ml on 3×3 cm) on waistline. Treatment was twice daily at 9 am and 4 pm (n=4 each).

[0238] FIG. 4: effect of treatment of rats with carbomer hydrogel (0.5 ml on 3×3 cm) followed by 5 mg/ml octopamine decanoate in carbomer hydrogel (0.5 ml on 3×3 cm) on plasma glycerol levels (n=4 each).

[0239] FIG. 5: potential synthesis route toward octopamine decanoate.

[0240] FIG. 6: Hydrolysis of 10 and 100 microM octopamine decanoate (FIGS. 6 a & b) and 10 and 100 microM synephrine decanoate (FIG. 6 c & d) in vitro at different concentrations of human fat.

[0241] FIG. 7: Hydrolysis of octopamine decanoate in plasma.

[0242] FIG. 8: Hydrolysis of octopamine decanoate, octopamine pentanoate and synephrine decanoate in PBS at 37 degrees.

[0243] FIG. 9: Stability of octopamine and synephrine in PBS at 37 degrees. Experiments are n=2.

[0244] FIG. 10: Inhibition of hydrolysis in fat suspension of octopamine decanoate 10 microM and synephrine decanoate 10 microM by beta antagonist propranolol (FIGS. 10a and c) and lipase inhibitor orlistat (FIG. 10 b and d). Experiments are n=4-8. The same data can be represented in time (FIG. 10e)

[0245] FIG. 11: In vitro skin penetration through human skin after application of 0.25 ml of octopamine hydrogel 2.75 mg/ml, octopamine decanoate 5 mg/ml and octopamine pentanoate 3.98 mg/ml. Penetration is concentration of 60 ml perfusion bath below the skin, 2 hrs after application. Experiments are n=2-3.

[0246] FIG. 12: Tissue concentration of octopamine in layers at increasing depth of human subcutaneous fat after application of 0.25 ml of octopamine (2.75 mg/ml), octopamine decanoate (5 mg/ml) and octopamine pentanoate (3.98 mg/ml), 20 hrs after application.

[0247] FIG. 13: Effect of Isoprenaline (FIG. 13a), octopamine (FIG. 13b), synephrine (FIG. 13c) and propranolol (FIG. 13d) on glycerol formation in a 250 mg/ml human fat suspension during 4 hrs at 37 degrees Celsius. Experiments are n=4-20.

[0248] FIG. 14: Effect of application on belly and hips of a hydrogel comprising 2.75 mg/ml free octopamine (2.5 ml, once daily), of a hydrogel comprising 5 mg/ml octopamine decanoate, (2.5 ml, once daily) and a hydrogel comprising 5 mg/ml octopamine decanoate (5 ml, twice daily) on waistline of humans. Experiments are the average of two experiments (n=2).

[0249] FIG. 15: Effect of application on belly and hips of control hydrogel without agonist or antagonist (2.5 ml, once daily), 2.75 mg/ml octopamine (2.5 ml, once daily), of 5 mg/ml octopamine decanoate (“OctDec”, 2.5 ml, once daily and 5 ml, twice daily) and 3.98 mg/ml octopamine pentanoate (“OctPent”, 2.5 ml, once daily) and of 5 mg/ml synephrine decanoate (“SynDec”, 2.5 ml, once daily and 5 ml, once daily) on waistline of a single individual.

[0250] FIG. 16: Effect of a hydrogel comprising 5 mg/ml octopamine decanoate (5 ml, twice daily) on belly and hip skinfold. Experiments are an average of 2 or 3 runs (n=2-3).

[0251] FIG. 17: Plasma levels of octopamine upon single administration of hydrogels comprising octopamine decanoate (5 mg/ml, 2.5 ml and 5 ml; FIG. 17a); free octopamine (2.75 mg/ml, 2.5 ml and 5 ml; FIG. 17b), and octopamine pentanoate (3.98 mg/ml 2.5 ml; FIG. 17c). Experiments are an average of one or two experiments (n=1-2).

[0252] FIG. 18: Plasma levels of free octopamine 12 hours after application, during chronic treatment with a hydrogel comprising 5 mg/ml octopamine decanoate (5 ml, twice daily). Experiments are an average of 1 or two runs (n=1-2).

[0253] FIG. 19: Systole, diastole and heart rate upon application of a hydrogel comprising 5 mg/ml octopamine decanoate (5 ml, twice daily) on belly and hips. Data represent an average of two persons.

[0254] FIG. 20: Effect of beta-3 agonists (SR 58611A, CL 316243, CGP12177), beta antagonist (SR 59230A), the alpha-2 antagonist Yohimbine (“Yo”), alpha 2 agonist Xylazine (“Xyl”) and phosphodiesterase inhibitor caffeine (“Cof”) on glycerol formation in a 250 mg/ml human fat suspension during 4 hrs at 37 degrees Celsius. Experiments are n=3-4.

[0255] FIG. 21: potential synthesis route toward synephrine decanoate.

[0256] FIG. 22: general synthetic approach toward prodrug esters of the invention, wherein R.sub.1a and R.sub.1b is H or OH, and at least one of R.sub.1a and R.sub.1b is OH, R.sub.2 is H or methyl, X is a leaving group, preferably chloride, bromide or iodide, R.sub.3 is benzyl or alkyl (preferably methyl or isopropyl), R.sub.4 is a C1-C31 alkyl group to provide the C2-C32 alkyl ester as defined above, and wherein at least of R.sub.5a and R.sub.5b is R.sub.4CO.

[0257] FIG. 23: General route for the synthesis of ester prodrugs, wherein 10 is an agonist or antagonist for an adrenergic receptor as defined elsewhere, which has a free OH-group, which free OH-group is preferably a benzylic or phenolic OH— group; and wherein 11 is an acylating agent, preferably an acid halide or an anhydride, wherein LG is a leaving group, preferably selected from a halide (preferably chloride), or a carboxylate, and wherein HM is a hydrolyzable moiety as defined elsewhere.

[0258] FIG. 24: A general route for formation of an amide prodrug 15, wherein 13 is an agonist or antagonist for an adrenergic receptor as described elsewhere, which has a free amine group comprising at least one amine hydrogen, which free amine group is preferably a primary alkyl amine, wherein R″ is selected from H or a C1-C8 linear, branched or cyclic alkyl group such as methyl, ethyl or isopropyl, preferably H, and wherein preferably, free OH-groups, more preferably phenolic or benzylic free OH-groups, are protected by a suitable protecting group, and wherein 14 is a hydrolyzable moiety as defined elsewhere functionalized with a carboxylic acid group, and wherein the coupling agent can be any known coupling agent, such as for instance DCC, EDCI, HATU or HBTU.

[0259] FIG. 25: A general route for formation of a carbamate prodrug 18, wherein 16 is an agonist or antagonist for an adrenergic receptor as described elsewhere, which has a free OH-group, which free OH-group is preferably a benzylic or phenolic OH— group; and wherein 17 is a hydrolyzable moiety as defined elsewhere, functionalized with an isocyanate.