Conjugates of VIP and active fragments thereof with hydrophobic moieties and topical compositions for use in the treatment of male impotence

Abstract: There are provided pharmaceutical compositions for topical transdermal application for the treatment of male impotence and compositions for the treatment of asthma and of hypertension. The active ingredients of such compositions are selected from vasoactive intestinal peptide (VIP), effective derivatives thereof and effective fragments thereof. In the compositions for the treatment of male impotence a transdermally effective conjugate of VIP, its derivatives or fragments coupled to a hydrophobic moiety, is used.

Description: The invention relates to pharmaceutical compositions for the treatment of male impotence. Impotence, a condition that can bring much suffering to the life of the afflicted individual and those surrounding him, can be caused by psychological as well as organic problems. It is estimated that in the United States alone, 10 million men suffer from varying degrees of impotence. In general, impotence is a predominant syndrome affecting at least 10-15% of the male population. In men above 40 years of age, the occurrence of impotence is prevalent due to neuroendocrine failures associated with aging, and any man over the age of 40 years may experience occasional impotence. The mechanism of penile erection is a complicated one and requires the intactness of the endocrine system, the nervous system and the vascular system. Many times the suffering individual is hesitant to seek medical help and at the moment the remedies available to the physician are rather limited. Organic abnormalities can be treated by surgery and implantations. Erection was also obtained by injection of smooth muscle relaxants such as phenoxybenzamine. It is, of course, quite understandable that superior treatment is needed and the invention relates to an ointment to treat impotence. It is the object of the invention to provide an improved product for the treatment of male impotence. This object is solved by the surprising finding that the transdermal application of vasoactive intestinal peptide (VIP) or derivatives or fragments thereof, coupled to a suitable hydrophobic moiety, enhances sexual activity. Therefore, the subject matter of the invention is a conjugate of vasoactive intestinal peptide (VIP), an active derivative or fragment thereof, coupled to a hydrophobic moiety and a transdermally effective pharmaceutical composition for topical application for the treatment of male impotence which comprises the conjugate as active ingredient. The active ingredients are VIP, functional derivatives thereof, active fragments thereof, and modified peptides wherein one or more of the amino acids are substituted by others. The backbone of the compositions is a naturally occuring material, or derivative thereof. The peptide of interests, Vasoactive Intestinal Peptide (VIP) fulfils several of the chemical criteria for a neurotransmitter in penile erection in the male: it is present in nerve fibers with nerve endings around cavernous smooth muscle and blood vessels and it is elevated during erection. Moreover, injection of exogenous VIP induces erection in man. It is important to add that in impotent men it was unequivocally shown that VIP quantities decrease locally in their penises. Since VIP was found to be a key neurotransmitter in erection formation, its local administration should help to relieve the dysfunction. Past studies used local VIP injection into the penis as the mode of drug administration. Using an animal model, we developed an ointment containing modified VIP which enhances mating behaviour in mammals, as evident from experiments with rats. The behavioural model for studies of sexual behaviour was the castrated rat model. Castrated rats lose their sexual activity in a time dependent manner and we used this characteristic to develop a model for investigating the therapeutic activity of VIP. The sequence of VIP, a 29-amino acid peptide, is as follows: The peptide chain was assembled according to the solid-phase methodology (Merrifield (1963) J. Am. Chem. Soc. 85:2149). Partial sequences were also synthesized by this approach. VIP₇₋₂₈ and VIP₁₆₋₂₈, for example, are active fragments. The following hydrophobic derivatives of VIP were prepared by attaching, as the last coupling step, a long chain aliphatic carboxylic acid to the N-terminus of the peptide chain. This was accomplished while the peptide was still attached to the polymeric support: 1. CH₃(CH₂)₆CO-VIP 2. CH₃(CH₂)₁₆CO-VIP 3. CH₃(CH₂)₁₆CO-VIP₇₋₂₈ 4. CH₃(CH₂)₁₆CO-VIP₁₆₋₂₈ 5. The following peptides were prepared by aminolysis of peptide-polymer conjungates with long-chain aliphatic amines; chain extension occurs at the C-terminus of the VIP: 1. VIP-NH CH₂CH₃ 2. VIP-NH(CH₂)₃CH₃ 3. VIP-NH(CH₂)₇CH₃ All peptides were purified by HPLC and characterized by their amino acid analysis. In general, a conjugate of VIP, or an active fragment thereof, said active fragment having the activity that injection of such fragment induces erection in a man, is coupled to a hydrophobic moiety. The hydrophobic moiety may be an aliphatic moiety, such as a moiety of the formula CH₃(CH₂) n -CO- where n is an integer from 1 to 16, or a moiety of the formula wherein n is an integer of from 2 to 6 and m is an integer of from 2 to 12, attached to the N-terminus of the VIP molecule, or an active fragment thereof, by coupling the corresponding carboxylic acids. The hydrophobic moiety may also be an aliphatic moiety, such as a moiety of the formula CH₃(CH₂) m -NH- wherein m is an integer of from 1 to 12, attached to the C-terminus of the VIP molecule, or an active fragment thereof, by coupling the corresponding amines. Examples Synthesis of Stearyl-VIP Synthesis was carried out via the solid-phase strategy. All amino acid derivatives were purchased from Peptide Institute Inc. (Japan). It was performed either automatically using Peptide Synthesizer (Applied Biosystem) or manually. α-Amino functional groups were protected by t-butyloxycarbonyl (t-Boc). Side chain protecting groups were as follows: His, N im -p-toluenesulfonyl; Lys, 2-chlorobenzyloxycarbonyl;p Tyr, 02.6-dichlorobenzyl; Arg, N G -p-toluenesulfonyl; Asp, β-benzyl; Thr and Ser, O-benzyl. The first amino acid (Asn) was attached to benzhydryl amine resin (Chemalog) using N.N'-dicyclohexylcarbodiimide (DCC) as a coupling agent. All coupling stages were performed with a threefold excess of protected amino acid derivatives using DCC and 1-hydroxylbenzotriazole (HOBt) as coupling agents. All stages of coupling were monitored with ninhydrin. The stearic acid residue was added similarly to amino acid residues using DCC/HOBt. Final cleavage of the peptide from the polymeric support along with deprotection was achieved with anhydrous HF. The product was first purified by gel-filtration on a Sephadex® G-25 column followed by HPLC on reversed phase C₁₈-column using a linear gradient obtained by mixing (A) 0.1% trifluoroacetic acid (TFA) in water and (B) 0.1% TFA in 70% acetonitrile and 30% water. The product gave, after acidic hydrolysis, the expected ratio of its constituent amino acids. Other peptides were similarly prepared. It is clear that introduction of hydrophobic moieties into the peptide chain of VIP can also take place within the chain, e.g., at the amino side chains of Lys. The hydrophobic moieties can be aliphatic, linear saturated (as in the compounds prepared), linear unsaturated (containing double or triple bonds), branched, saturated and unsaturated, linear and branched containing heteroatoms, aromatic-aliphatic or aromatic, and can be introduced into either C, N terminus or within the peptide chain, or at two or more positions. All these modifications can be performed on fragments of VIP as well, or on any other biologically active analog of VIP. The animal model All tested rats (Wistar derived from the departmental colony, Hormone Research Department, at the Weizmann Institute of Science), are sexually experienced male rats around 3 months of age (body weight ∼250 g). At this age, the rats are castrated by removal of the testes and injected daily with testosterone for at least 14 consecutive days. All rats (males and females) that take part in the experiment are kept in a 12-hour light 12-hour dark cycle. Prior to testing, each male is put in a separate cage for at least 1 hour. A sexually receptive female is introduced to each male. Females were tested for their receptivity by vaginal smearings. The latencies of mounts and intromissions are recorded. The tests are terminated if the male does not mount within 15 minutes. Using castrated animals we achieved decreased sexual activity which is expressed by longer intervals between intromissions and less intromissions during the 15 min observation periods. Initially, we tested a few VIP analogs by injection (intravenously 5-10 µg/animal). As a control we injected saline at identical volumes (5-10 µl). Table I shows the results for stearyl-VIP injection. At the upper left side of the chart we see that stearyl-VIP potentiates sexual activity in a comparable manner to VIP (right hand side of the table). This analog is lipophylic and hence may be a candidate for topical application. As we have obtained positive results with stearyl-VIP by injection we have now developed a topical application method. This was achieved by mixing 20 µl of 1 µg/ml VIP-stearyl with 10 µl dimethyl sulfoxide (DMSO) and direct topical application on the sex organ. In Table II one can easily notice a remarkable sexual arousal after stearyl-VIP application compared with control rats subjected to DMSO. We therefore suggest that stearyl-VIP could be used as a potentiator of sexual activity in animals. In Table III we compare the topical application of the carrier (DMSO) to stearic acid by itself, to VIP by itself and to stearyl-VIP. It is self-evident that stearyl-VIP is the most active analog. A shorter (8 amino acid) VIP analog which is related to the human immunodeficiency virus (HIV) peptide T (HIV-PEPT) which did not show much activity upon topical application by itself is somewhat active in a conjugated form but not as VIP-stearyl. (A)The effect of topical application of stearyl-VIP on the sexual behaviour of male rats is summarized in Table IV. Preparation of Ointment - Lanolin (1 g), water (3 g) DMSO (7 drops) and 10 mg of stearyl-VIP were manually homogenized in a mortar. An amount of paste, containing 30-50 µg peptide was applied onto the rat's penis and sexual behaviour, following the methodology previously described, was monitored. As shown in the Table, the peptide showed about 25% decrease in intromission latency and two-fold increase in ejaculations in 10 animals. (B) Using the same protocol as in (A) an ointment of VIP-oleyl was prepared and tested. It did show a certain effect in shortening of intromission latency (∼10%) and also increased, though much less than stearyl-VIP, the number of ejaculations. The effective dose for humans is between 1 and 5 µg. We claim that VIP-stearyl can be used in topical application to improve mammalian sexual behaviour in sexually deficient males. Moreover, we claim that further modification using the same idea of a lypophylic derivative may even improve the material as VIP is known to be a bronchodilator as well as a vasodilator. Other therapeutical applications such as in asthma and blood pressure regulation are also claimed. Legends to tables Table I Rats were castrated and injected daily for 15 consecutive days with testosterone at 4 µg/100 g body weight (BW). Tests were performed with stearyl-VIP or VIP at 10 µg/animal. Table II Rats were castrated and injected daily for 16 consecutive days with testosterone at 2 µg/100 g BW. Testing was performed by topical application of stearyl-VIP-20 µg/20 µl mixed with 10 µl DMSO and compared to topical application of 30 µl DMSO. Table III 6 rats, 120 days old, sexually experienced, were tested. The rats were castrated 14 days earlier and were injected with 4 µg/100 g BW of testosterone daily. In the table we see an increase in number of intromissions and shortening of latency after topical application of HIV-PEPT-stearyl and stearyl-VIP. All 6 materials were tested by topical application. Also there is a clear increase in the number of ejaculations after applications of HIV PEPT-stearyl and stearyl-VIP. Corresponding results are expected with human males. Table IV Summary of the effect of topical application of stearyl-VIP on the sexual behaviour of male rats. TOPICAL APPLICATION Rat DMSO Stearyl-VIP Obs. Intromission latency Obs. Intromission latency A₁ 11 48±12 21 23±4 A₂ 8 79±30 14 29±4 A₃ - - 14 32±8 B 21 31±5:E 10 16±3:E 3 38±10 B₂ - - 11(3M) 18±7 B₃ 12 54±12 25 24±4 Please rote that rats B₂ and A₃ were sexually active only after Stearyl-VIP application.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, FR, GB, GR, IT, LI, LU, NL, SE A conjugate of vasoactive intestinal peptide (VIP), or of an active fragment thereof, said active fragment having the activity that injection of such fragment induces erection in a man, coupled to a hydrophobic moiety. A conjugate according to claim 1 wherein the hydrophobic moiety is an aliphatic moiety or a moiety of the formula wherein n is an integer of from 2 to 6 and m is an integer of from 2 to 12, and is attached to the N-terminus of the VIP molecule or an active fragment thereof. A conjugate according to claim 1 or 2 wherein a hydrophobic moiety is a moiety of the formula CH₃(CH₂) n -CO-, where n is an integer of from 1 to 16, attached to the N-terminus of the VIP molecule or an active fragment thereof. A conjugate according to claim 1 wherein the hydrophobic moiety is a moiety of the formula CH₃(CH₂) m -NH-, wherein m is an integer of from 1 to 12, attached to the C-terminus of the VIP molecule or an active fragment thereof. The conjugate of any one of claims 1 to 4 wherein the active fragment of VIP is VIP₇₋₂₈ or VIP₁₆₋₂₈. The conjugate of claim 1 wherein the conjugate is represented by the formula CH₃(CH₂)₆-CO-VIP or wherein the hydrophobic moiety is coupled through the N-terminus of the VIP, or active fragment thereof, or VIP-NHCH₂CH₃ VIP-NH(CH₂)₃CH₃ or VIP-NH(CH₂)₇CH₃ wherein the hydrophobic moiety is coupled through the C-terminus of the VIP. The conjugate of claim 1 having the formula A transdermal pharmaceutical composition useful for the treatment of male impotence comprising a physiologically effective quantity of a conjugate according to any one of claims 1 to 7 combined with a pharmaceutically acceptable carrier. A composition according to claim 8 wherein the conjugate is stearyl-VIP and the carrier comprises DMSO. A process for the preparation of a conjugate of vasoactive intestinal peptide (VIP), or an active fragment thereof, said active fragment having the activity that injection of such fragment induces erection in a man, coupled to a hydrophobic moiety, which comprises coupling a long chain aliphatic carboxylic acid to the N-terminus of the peptide chain or coupling a long-chain aliphatic amine to the C-terminus of the peptide chain. The process of claim 10 wherein the aliphatic carboxylic acid is CH₃(CH₂)₁₆-COOH and the conjugate is one having the formula: The process of claim 10 or 11 wherein the coupling of the aliphatic carboxylic acid to the N-terminus of the peptide chain comprises coupling stearic acid to the N-terminus of the peptide chain using N,N'-dicyclohexyl-carbodiimide and 1-hydroxylbenzotriazole as coupling agents. Claims for the following Contracting State : ES A process for the preparation of a conjugate of vasoactive intestinal peptide (VIP), or an active fragment thereof, said active fragment having the activity that injection of such fragment induces erection in a man, coupled to a hydrophobic moiety, which comprises coupling a long chain aliphatic carboxylic acid to the N-terminus of the peptide chain or coupling a long-chain aliphatic amine to the C-terminus of the peptide chain. The process according to claim 1 wherein the aliphatic carboxylic acid has the formula CH₃(CH₂) n -COOH, where n is an integer from 1 to 16. The process according to claim 1 wherein the aliphatic amine is an amine of the formula CH₃(CH₂) m NH₂, wherein m is an integer from 1 to 12. The process of any one of claims 1 to 3 wherein the active fragment of VIP is VIP₇₋₂₈ or VIP₁₆₋₂₈. The process of claim 1 wherein the aliphatic carboxylic acid is CH₃(CH₂)₆-COOH and the conjugate is CH₃(CH₂)₆CO-VIP. The process of claim 1 wherein the aliphatic carboxylic acid is CH₃(CH₂)₁₆-COOH and the conjugate is one having the formula: The process of any one of claims 1, 2, 4 or 6 wherein the coupling of the aliphatic carboxylic acid to the N-terminus of the peptide chain comprises coupling stearic acid to the N-terminus of the peptide chain using N,N'-dicyclohexylcarbodiimide and 1-hydroxylbenzotriazole as coupling agents. A process for the preparation of a transdermal pharmaceutical which comprises combining a physiologically effective quantity of a conjugate prepared according to any one of claims 1 to 7 with a pharmaceutically acceptable carrier. The process of claim 8 wherein the conjugate is stearyl-VIP and the carrier comprises DMSO.

Inventors: FRIDKIN MATITYAHU, GOZES ILLANA, FRIDKIN, MATITYAHU, GOZES, ILLANA
Applicants: YEDA RES & DEV, YEDA RESEARCH AND DEVELOPMENT COMPANY LIMITED
Application year: 1989
Main classification: A61K38
IPC codes: A61K38, A61P15, C07K14, A61K9, C07K1, A61P13