WO1994027629A1

WO1994027629A1 - Method to control appetite and treat obesity

Info

Publication number: WO1994027629A1
Application number: PCT/US1994/005177
Authority: WO
Inventors: Jonathan A. Tobert
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1993-05-28
Filing date: 1994-05-10
Publication date: 1994-12-08
Anticipated expiration: 1995-11-28
Also published as: AU7242394A

Abstract

The present invention relates to the administration of human apolipoprotein A-IV, isoforms and active fragments thereof, to humans as an injectable formulation for the purpose of controlling appetite and reducing food intake and body weight. The invention is also directed to an injectable pharmaceutical formulation of human apolipoprotein A-IV, or active fragments thereof.

Description

TITLE OF THE INVENTION

METHOD TO CONTROL APPETITE AND TREAT OBESITY

SUMMARY OF THE INVENTION

BACKGROUND OF THE INVENTION

Obesity is a ubiquitous problem in industrialized countries and can produce major medical and psychosocial liabilities. Obesity is a difficult medical problem because there are no adequate methods available for long term appetite control. Dietary therapy generally fails to produce sustained weight loss and the available drugs-sympatheo- mimetic amines and agents increasing brain serotonin levels— are not currently indicated in the United States for long-term treatment of obesity. Consequently, the treatment of obesity generally meets with limited success. Eating behavior is difficut to modify, except by surgical means. Patients with severe obesity sometimes undergo surgical procedures such as jaw wiring and gastric stapling to control their appetite and reduce their weight. The present invention fulfills the need for a safe and effective non-surgical treatment of obesity.

A problem related to obesity is bulimia nervosa, a behavioral disease characterized by excessive "binge" eating followed by vomiting or other purging behavior. The method of the present invention controls appetite and may be useful in the treatment of bulimia nervosa.

It appears that each individual has a barometric "set point", i.e., a body weight he or she will maintain over long periods of time if food supplies are adequate. The set point is determined by genetic, familial and cutural factors. For most individuals, body weight is determined mainly by eating behavior, with other factors such as basal metabolic rate and energy expenditure by exercise playing a more minor role. Experiments on parabiotic rodents have suggested the existence of one or more long-acting circulating satiety factors, which suppress appetite and consequently food intake.

The identity of this appetite suppressing factor or factors has remained obscure until very recently. Fujimoto et al., "Suppression of Food Intake by Apolipoprotein A-IV is Mediated through the Central Nervous System in Rats" J. Clin. Invest. 9∑: 1830 (April, 1993); Fujimoto et al-, "Increased apolipoprotein A-IV in rat mesenteric lymph after lipid meal acts as a physiological signal for satiation" Am. J. Phvsiol. 262: G1002 (1992) and Fujimoto et l-, "Effect of intravenous administration of apolippoprotein A-IV on patterns of feeding, drinking and ambulatory activity of rats" Brain Research 608: 233 (1993) show food intake in rats may be suppressed by apolipoprotein A-IV. The function of this apolipoprotein of molecular weight about 45,000 was previously undefined. It is known to be secreted by the small intenstine following the ingestion of fat and is probably catabolized by the liver. Unlike other apolipoproteins, a large fraction of circulating apolipoprotein A-IV is lipoprotein-free or only loosely associated with lipoproteins, making it potentially transportable across the blood brain barrier.

The plasma half life of human apolipoprotein A-IV is several hours. The level of this glycoprotein in blood (and hence in the brain satiety center) is determined by secretion by the gut, which is known to be stiumulated by ingestion of fat, and probably by catabolism by the liver. Little is known about the latter; it is known that apolipo¬ protein A-IV binds to hepatocellular membranes and thus is probably taken up and metabolized by the liver. It is likely that the liver, as the central organ of metabolism, plays a major role in the control of body weight, including bringing an organism to its "set point."

There are presently four known isoforms of human apolipoprotein A-IV. An isoform is a genetically determined variant of a protein. Depending on the population examined, the allele frequency of human apolipoprotein A-IV-1, the primary isoform, is about 94%; for apolipoprotein A-IV -2, the second most prevalent isoform, the allele frequency is about 6%. Apolipoprotein A-IV-3 and A-IV -4 are the other identified isoforms; the allele frequency for these isoforms is very low. See Menzel et ah, J. Lipid Res. 23: 915-922 (1990) and Lohse et al.. J. Biol. Chem. 265: 12734-12739 (1990).

Fujimoto et al. suggest that apolipoprotein A-IV may be a long acting satiety factor in rats; they show that apolipoprotein A-IV concentrations in cerebrospinal fluid increase during feeding, that infusion of apolipoprotein A-IV into the third ventricle inhibits food intake in fasted animals and that feeding behavior can be stimulated by antibodies to apolipoprotein A-IV infused into the third ventricle.

The present invention surprisingly provides that human apolipoprotein A-IV, isoforms thereof and active fragments thereof, can be used to control appetite in humans. No apolipoproteins or lipoproteins have been used for any accepted therapeutic purpose to date.

The present invention relates to the administration of human apolipoprotein A-IV, and active fragments thereof, to humans as injectable formulations for the purpose of controlling appetite and reducing food intake and body weight, and controlling obesity and bulimia. The invention is also directed to an injectable pharmaceutical formulation of human apolipoprotein A-IV or an active fragment thereof and an autoinjection device for administration of human apolipoprotein A-IV and active fragments thereof. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to administration of human apolipoprotein A-IV, isoforms and active fragments thereof, to humans as an injectable formulation, preferably a subcutaneous formulation, for the purpose of controlling appetite and reducing food intake and body weight.

Human apolipoprotein A-IV is a 47 kD glycoprotein which is secreted by the intestine in chylomicrons and, unlike other apolipoproteins, circulates partly free and partly loosely associated with high density lipoprotein. Its regulation and function are largely obscure.

Human apolipoprotein A-IV may be produced by recombinant DNA technology and expressed in an expression system such as I coli. However, the entire glycoprotein of molecular weight 47 kD need not be expressed; active fragments of human apolipoprotein A-IV, may also be employed. An active fragment of human apolipoprotein A-IV is defined for purposes of the present invention as an expressed fragment of human apolipoprotein A-IV having appetite suppressant activity. The appetite suppressant activity of a fragment may be measured by intravenous infusion of the fragment of apolipoprotein A-IV over 2 to 8 hours at doses ranging from 10 to 1000 μg/min. in fasted healthy human volunteers with placebo control. Appetite suppressant activity is measured by a visual analog scale and a questionnaire, and by reduction of consumption of offered standard food items. A fragment that reduces appetite and/or food consumption over the infusion period by 20% or more is an active fragment of apolipoprotein A-IV for the purposes of the present invention.

The cloning, expression and purification of human recombinant apolipoprotein A-IV is known in the art. This procedure is described in Du verger et ah, "Functional characterization of human recombinant apolipoprotein A-IV produced in Escherichia coli" Eur. J. Biochem. 201(2): 373-83 (1991); Philit et al, DNA and Cell Biol. 9: 129-137 (1990); Denefle et al., Gene 85: 499-510 (1989). Elshourbagy et al, J. Biol. Chem. 261 : 1988-2002 (1986) discloses a nucleotide sequence for the human apolipoprotein A-IV. Fragments of human apolipoprotein A-IV may be expressed and purified according to procedures known in the art.

The present invention is also directed to a method of controlling appetite which comprises the administration to a human subject in need of such treatment of a nontoxic therapeutically effective amount of human apolipoprotein A-IV, including isoforms thereof, or an active fragment thereof, by injection. Specifically, the method of this invention is useful in treating obesity, bulimia nervosa and like diseases in humans for which treatment requires reduction in food intake or suppression of appetite. The human apolipoprotein A-IV, or active fragment thereof, may be administered subcutaneously or intramuscularly, preferably subcutaneously since subcutaneous administration causes less patient discomfort. Administration may occur one to four times daily or less frequently at a therapeutically effective dose. The human apolipoprotein A-IV or an active fragment thereof may also be administered on an intermittent basis when therapeutically advisable, e.g. in the treatment of bulimia nervosa. Dosages may be varied, depending on the age, severity of obesity and other conditions of human patients, but daily dosage for adults is within a range of from about 0.1 mg to 2000 mg, preferably 1 mg to 500 mg, which may be given in a single dose or two or more divided dosages. Higher dosages may be favorably employed as required.

Human apolipoprotein A-IV, or an active fragment thereof produced by recombinant DNA technology or other means is formulated as an injectable formulation, preferably a subcutaneous injection. The formulation may be formulated as either a suspension or a solution. A suspension may have the advantage of providing more gradual release of the active agent. The suspensions or solutions of the formulation of the present invention contain 0.1% to 15% human apolipoprotein A-IV, preferably 1% to 10%, in a pharmaceutically acceptable carrier.

The human apolipoprotein A-IV is administered as an injectable suspension or solution comprising human apolipoprotein A- IV, or an active fragment thereof, and a suitable pharmaceutical carrier. These injectable suspensions may be formulated according to known art, using suitable non-toxic, parenterally acceptable diluents or solvents, such as 1 ,2-propanediol, water, Ringer's solution, dextrose solution or isotonic sodium chloride solution. These injectable suspensions or solutions may further contain excipients suitable for manufacture of aqueous suspensions and solutions. Such excipients may be:

(1) suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;

(2) dispersing or wetting agents which may be:

(a) a naturally occurring phosphatide such as lecithin,

(b) a condensation product of any alkylene oxide with a fatty acid, for example, polyoxyethylene stearate,

(c) a condensation product of an ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol,

(d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyyoxyethylene sorbital monooleate, or

(e) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol anhydride, for example, polyoxyethylene sorbital monooleate. Such suspensions and solutions may further contain microcrystalline cellulose for imparting bulk and methylcellulose as a viscosity enhancer.

The aqueous suspensions and solutions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxy- benzoate, and antioxidants and the like may be incorporated as required.

In addition, the aqueous suspension or solution may, if necessary, be buffered to a physiologically appropriate pH by the addition of a suitable buffer such as sodium acetate, sodium lactate, sodium benzoate or Tris.

Dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients may also be present.

An oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example: arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example: beeswax, hard paraffin or cetyl alcohol. These compositions may be prepared by the addition of an antioxidant such as ascorbic acid.

It is preferred that the human apolipoprotein be formulated in a suspension.

The pharmaceutical compositions of the suspensions and solutions of human apolipoprotein A-IV, and active fragments thereof, of the present invention when administered by intramuscular or subcutaneous injection avoid the inconvenience of intravenous administration while maintaining the advantages of parenteral administration, i.e,, good bioavailability. This treatment, although it requires an injection, is likely be accepted by patients with moderate to severe obesity. (Surgical procedures such as jaw wiring and gastric stapling are currently employed in some severely obese patients.) In addition, the formulation of the present invention may be used in conjunction with modern auto-injection devices which increases the convenience and decreases the discomfort of subcutaneous injections. The use of the formulation of the present invention with such autoinjection devices may increase patient compliance by improving ease of administration.

The following example is given for the purpose of illustrating the present invention and shall not be construed as being limitations on the scope or spirit of the invention.

EXAMPLE 1

Formulation of human apolipoprotein A-IV An injectable formulation is prepared as follows

Human apolipoprotein A-IV 10 mg

Polysorbate 80 10 μg

Sodium chloride 10 mg

Mannitol 10 mg

Distilled water for injection q.s. per vial of 1 mL

The formulation is filtered to remove organisms and dispensed into a 2 mL glass vial.

EXAMPLE 2

One hundred and eleven hypocholesterolemia patients participated in a four period cross-over study published in Hunninghake et al-. "The Efficacy of Intensive Dietary Therapy Alone or Combined with Lovastatin in Outpatients with Hypercholesterolemia" New England Journal of Medicine 328: 1213-1219 (1993). The published study investigated the lipid-lowering efficacy of a diet low in saturated fat and cholesterol, with and without lovastatin, an HMG-CoA reductase inhibitor. Briefly, at the end of the nine week study periods the diet lowered LDL and HDL cholesterol by 5 and 6%, respectively , and did not affect fasting VLDL cholesterol or plasma triglyceride levels.

The present experiment involved assaying apolipoprotein A-IV in archived specimens from the above-described study, using a sandwich ELISA method.

There was sufficient EDTA plasma for assay in 256 of the original 405 samples, which had been collected after a 12h overnight fast and stored at -70°C for 4-5 years. To test for degradation, apolipoprotein A-I was also assayed in the stored specimens, the correlation coefficient with the original measurements was 0.98, suggesting that storage conditions were adequate. The data were analyzed by a general linear model technique as in the original report, using the per-protocol approach which eliminated data from study periods in which adherence to the diets fell outside pre-specified limits.

Table 1: Mean (95% C.I.) values of apolipoprotein A-IV and body weight at the end of the 9 week periods, with corresponding values for reported dietary energy and fat content.

TABLE 1

Variable High Fat/ Low Fat/ High Fat/ Low Fat/ P Value P Value Placebo Placebo Lovastatin Lovastatin For Diet For Drug

N=51 N=55 N=57 N=52

Apo A-IV 6.39 4.91 6.18 5.32 <0.001 0.8

(5.82-6.96) (4.44-5.38) (5.63-6.73) (4.81 -5.83)

N=85 N=84 N=84 N=82 o

I energy 2280 1570 2230 1550 (kcal) (2150-2410) (1470-1670) (2110-2350) (1440-1660)

% fat 41.7 24.8 41.8 24.2 (40.9-42.5) (23.6-25.0) (41.0-42.6) (23.0-25.4)

body weight(kg) 74.7 73.9 73.9 72.8 <0.001 0.4

(71.9-77.5) (71.0-76.8) (71.0-76.8) (70.1-75.5)

As shown in Table 1 , lovastatin had no effect on plasma apolipoprotein A-IV, but the low-fat diet produced a substantial and significant decrease. As previously discussed, the differences in dietary composition (though obtained by standard diary and analysis techniques) are probably overestimated, as suggested by the modest changes in weight (as well as lipids).

Figure 1 shows the relative composition and effects of the low- fat diet on all the apolipoproteins measured. Since there was no evidence for an interaction between the dietary and drug interventions for apolipoprotein A-IV or any other apolipoprotein, the overall percent difference in the mean for the low fat diet compared to the high fat diet are shown. (Similar results were obtained when the lovastatin periods were excluded.)

Figure 1 : Percent Difference In Mean Apolipoprotein Levels, Low Fat Versus High Fat Diet. Error bars represent 95% C.I.

Apolipoprotein

A-l A -II A-IV B

-5

φ υ -10 c Φ

Φ -15

G a? -20

-25

-30 It is clear that the effect of the diet was relatively specific for apolipoprotein A-IV, for which the change in the mean was 20%, compared to 6% or less for all the other apolipoproteins assayed. As with the other variables measured, there was no evidence from an analysis of interaction to suggest that the apolipoprotein A-IV response to diet was different in obese patients.

This study conclusively demonstrated that apolipoprotein A-IV is higher in patients consuming a high fat diet compared to those on a low- fat diet.

While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the casual variations, adaptations, modifications, deletions, or additions of procedures and protocols described herein, as come within the scope of the following claims and its equivalents.

Claims

WHAT IS CLAIMED IS:

1. An injectable pharmaceutical composition comprising: human apolipoprotein A-IV or an active fragment thereof and a pharmaceutically acceptable carrier.

2. The injectable pharmaceutical composition of Claim 1 comprising 0.1 % to 15% (w/v) of human apolipoprotein A-IV.

3. The injectable pharmaceutical composition of Claim 1 comprising 0.1% to 15% (w/v) human apolipoprotein A-IV and a pharmaceutically acceptable carrier.

4. The injectable pharmaceutical composition of Claim 1 wherein the carrier is comprised of polysorbate 80 and aqueous sodium chloride.

5. The injectable pharmaceutical composition of Claim 1 wherein the carrier is comprised of sodium carboxymethylcellulose and aqueous sodium chloride.

6. The injectable pharmaceutical composition of Claim 1 wherein the carrier is comprised of aqueous sodium chloride.

7. The injectable pharmaceutical composition of Claim 1 which additionally comprises sodium carboxymethylcellulose and sodium acetate.

8. The injectable pharmaceutical composition of Claim 1 which additionally comprises 2-amino-2-hydroxymethyl-l,3-propane diol and sodium chloride.

9. A method of treating obesity in a human patient comprising the administration by injection of a therapeutically effective amount of human apolipoprotein A-IV or an active fragment thereof to the patient.

10. The method of treating obesity in a human patient of Claim 9 comprising the administration by intramuscular injection of a therapeutically effective amount of human apolipoprotein A-IV or an active fragment thereof to the patient.

11. The method of treating obesity in a human patient of Claim 9 comprising the administration by subcutaneous injection of a therapeutically effective amount of human apolipoprotein A-IV or an active fragment thereof to the patient.

12. The method of treating obesity in a human patient of Claim 9 comprising the daily administration by injection of 0.1 mg to 2000 mg of human apolipoprotein A-IV or an active fragment thereof to the patient.

13. The method of treating obesity in a human patient of Claim 9 comprising the daily administration by injection of 1 mg to 500 mg of human apolipoprotein A-IV or an active fragment thereof to the patient.

14. The method of treating obesity in a human patient of Claim 9 wherein the human apolipoprotein A-IV is administered from one to three times daily.

15. The method of treating obesity in a human patient of Claim 9 wherein the human apolipoprotein A-IV is administered with an autoinjection device.

16. A method of treating bulimia nervosa in a human patient comprising the administration by injection of a therapeutically effective amount of human apolipoprotein A-IV or an active fragment thereof to the patient.

17. The method of treating bulimia nervosa in a human patient of Claim 16 comprising the administration by subcutaneous injection of a therapeutically effective amount of human apolipoprotein A-IV or an active fragment thereof to the patient.

18. The method of treating bulimia nervosa in a human patient of Claim 16 comprising the daily administration by injection of 0.1 mg to 2000 mg of human apolipoprotein A-IV or an active fragment thereof to the patient.

19. The method of treating bulimia nervosa in a human patient of Claim 16 comprising the daily administration by injection of 1 mg to 500 mg of human apolipoprotein A-IV or an active fragment thereof to the patient.

20. The method of treating bulemia nervosa in a human patient of Claim 16 wherein the human apolipoprotein A-IV is administered from one to three times daily.