Patent Application
20070275901
The present invention relates to the synthesis, modification and reduction of the primary structure of arterial hypotension inducing peptides (hypotensive peptides) named Tityus serrulatus Hypotensins I to IV found in yellow scorpion Tityus serrulatus.
Hypertension is an important challenge for the international public health authorities due to its high occurrence in modern society, in addition to the cardiovascular and renal risks derived therefrom. It is estimated that approximately 26% of the world population, that is, 972 million people suffered from hypertension in year 2000.
Blood pressure, or arterial pressure, is measured as systolic (pressure of the blood in the arteries when the ventricular systoles occurs) and diastolic (when diastole occurs). According to World Health Organization (WHO) the optimal blood pressure is less than 120 systolic and 80 diastolic (120/80 mmHg). High blood pressure, or hypertension, is considered to be a pressure greater than or equal to 140/90 mmHg and “high normal” blood pressure is between 130/85 and 140/90 mmHg (Bulletin of the World Health Organization, 1999).
Hypertension may be classified as primary and secondary. Primary or essential hypertension does not have a specific cause, being the most common form of hypertension and being presented by 95% of the patients. Genetic factors seem to be one of the major causes of this kind of hypertension.
The causes of secondary hypertension are known and pregnancy, cirrhosis and renal disorders may contribute for a temporary hypertensive condition. Some medicaments such as cortisone and estrogen may also temporarily increase the blood pressure. Prolonged use of anti-inflammatory drugs (NSAIDs) such as aspirin may cause renal disorders and can also affect the treatment of individuals suffering from hypertension who make use of anti-hypertensive drugs such as β-blockers and diuretics. It is known that cocaine causes acute hypertension conditions although apparently it does not cause a chronic condition. A study shows that about 10% of the cases related to hypertension are caused by the abuse of alcohol ingestion. Caffeine causes a temporary increase of blood pressure. However, studies show that its regular consumption increases the risks of heart diseases in health individuals. The dangers associated with caffeine, however, cannot be compared to smoking, which may increase the risk of death by cardiomyopathy and hypertension. Blood pressure may also temporarily increase due to stress or physical activities.
At the present time, five classes of drugs are recognized as efficient for hypertensive treatment: diuretics, alpha-blockers, beta-blockers, angiotensin converting enzyme (ACE) inhibitors, calcium antagonists, and angiotensin II antagonists.
Diuretics cause the body to excrete water and salt, thus decreasing the blood plasma volume and, consequently, decreasing the blood pressure. ACE inhibitors reduce the production of angiotensin-II and reduce the degradation of bradykinin. Beta-blockers inhibit the increase of the heart rate caused by the exciting effect of noradrenaline. Vasodilators expand blood vessels and calcium channel blockers help decrease the contractions of the heart.
All drugs used for hypertension have side effects and, therefore, there is still a need for novel anti-hypertensive drug classes.
The use of peptides as active ingredients for medicaments is a promising application of biotechnology. The major advantages rely on the selectivity and affinity of those molecules, as well as on the possible decrease of side effects and toxicity that result from chemical intermediates and/or metabolites. There are, on the other hand, important drawbacks that must be overcome for using those molecules such as their size that has influence on their absorption and distribution, as well as the resistance thereof against enzymatic hydrolysis and degradation in vivo.
Presently known anti-hypertensive peptides are isolated from animal venoms and have a primary structure comprising from 5 to 13 amino acid residues. Such peptides have anti-hypertensive activity based on the inhibition of angiotensin converting enzyme (ACE) that prevents the hydrolysis of bradykinin (a hypotensive agent) and the synthesis of angiotensin-II (a hypertensive agent), thereby acting as anti-hypertensive peptides.
Scorpion Hypotensive Peptides (SHptP) named TsHpt-I, TsHpt-II, TsHpt-III and TsHpt-IV, which are disclosed in patent application U.S. Ser. No. 10/517,097 refer to novel anti-hypertensive peptides found in animal origin venom. They i) consist of 24 to 25 amino acid residues, not crosslinked by disulfide bridges (absence of cysteine) and having molecular weights in the range of 2500 to 3000 Da; ii) have a molecular signature with amino acid residues Pro-Pro or Pro-Pro-Ala in their carboxy-terminal ends; and iii) induce a potent, long and sustained arterial hypotension, with one of the action mechanisms thought to be the potentialization of bradykinin occurring independently from the inhibition of the angiotensin converting enzyme.
The present invention relates to synthetic peptide primary structures comprising an amino acid sequence selected from the group of SEQ ID NOs:1 to 12, wherein said primary structure comprises an +aa-Pro-Pro amino acid in which “+aa” is Lys, Arg, His or any other modified amino acid that has a positive charge at physiological pH.
In another embodiment, the present invention also relates to a recombinant peptide primary structure as defined above prepared by recombinant techniques in heterologous expression systems such as viral systems, bacterial systems, fungal systems or in any other expression systems in eukaryotic or prokaryotic cells, or combinations thereof.
Still in accordance with another embodiment the present invention relates to a pharmaceutical composition comprising at least one peptide having a primary structure as defined above.
The present invention also relates to a method for treating hypertension, comprising administering to an animal at least one peptide of the invention.
The invention will be described in conjunction with the following drawings wherein:
In order to reduce the native structure of the hypotensive peptides TsHpt-I, TsHpt-II, TsHpt-III and TsHpt-IV, the inventors developed a synthesis based on the Fmoc solid phase technique (see, e.g., CHAN, W. C & WHITE, P. D. “Fmoc solid-phase peptide synthesis. A practical approach.” Oxford University Press; 2000) to synthesize peptides analogous to TsHpt, the structures of which were reduced to a minimum functional structure. Such minimization aims also to decrease production costs, to optimize their absorption and distribution of the active ingredient in the individual organism and to optimize their protection against enzymatic hydrolysis and degradation in vivo.
As the native TsHpts have a structure with 24 or 25 amino acid residues the primary sequence of which has 2 proline residues in its carboxy-terminal portion, the inventors developed a synthesis of peptides analogous to the native molecules based on their carboxy-terminal portion and obtained the following synthetic amino acid sequences 1 to 12:
The above peptide primary structures of the invention comprise +aa-Pro-Pro amino acids wherein “+aa” is Lys, Arg, His or any other modified amino acid that has a positive charge in physiological pH.
The insertion of chemical modifications such as acetylation, cyclization, amidation, amongst others, in the amine- and carboxy-terminal ends and/or in the side chains have the aim of providing a higher level of protection of the primary structure against enzymatic hydrolysis and degradation that may increase the active ingredient plasmatic half-life leading to a longer pharmacological effect.
The hypotensive effect of the synthetic analogues represented by SEQ ID NOs:1 to 12 as well as native peptide TsHpt-I demonstrate by in vivo assays a capacity for inducing hypotension independently from the addition of bradykinin and from the inhibition of angiotensin converting enzyme. Those effects could be observed through, for example, the intravenous injection of those compounds in normotensive Wistar rats, that is, rats having blood pressure at acceptable levels, at doses varying in a range of 35 to 43 μg/kg as shown in
In the normotensive rats the intravenous injection of both native and synthetic peptides led to an immediate decrease of the mean arterial pressure of 5 to 20 mmHg independently from the addition of bradykinin. The decrease of the mean arterial pressure due to administration of bradykinin (bradykinin potentializing effect) in those cases remains for more than 120 minutes.
In the rats of hypertensive strains (STR and TGR(mRENs)27) the mean arterial pressure decrease induced by intravenous injection of synthetic peptides, independently from administration of bradykinin, was measured for 6 hours with values varying from 5 to 35 mmHg. In vitro assays using preparations with aortic vessels isolated from male Wistar rats showed that synthetic peptides are capable of inducing vasodilatation dependent from production of nitric oxide, which explains its hypertensive effect (see
Hypotensive effects independent from bradykinin and from inhibition of ACE as those observed for native peptide TsHpt-I for its synthetic analogues, confirm a enhanced pharmacological action in relation to other bradykinin potentializing peptides identified, isolated and studied up to this moment and place those new peptides in a new class of hypotension inducing peptides present in animal venoms.
It is known that one of the major problems for developing proteic drugs is their low stability in the gastrointestinal tract and their poor absorption by the intestinal cells. However, the synthetic peptide of SEQ ID NO:5, for example, was able to induce a prolonged reduction of blood pressure after oral administration at a dose of 2.5 mg/Kg of rat body weight), demonstrating that the molecule is resistant to the action of proteolytic enzymes present in the gastrointestinal tract, in addition to being properly absorbed in the mucosa thereof.
The peptide primary structures of the present invention may be prepared by recombinant techniques in heterologous expression systems such as viral systems, bacterial systems, fungal systems or in any other expression systems in eukaryotic or prokaryotic cells, or combinations thereof.
The peptide primary structures of the present invention are useful as active ingredients in pharmaceutical compositions.
Peptide Synthesis (Fmoc Route) and Chemical Modification of Peptides
Peptides were synthesized using the Fmoc/t-butyl route of synthesis on a solid support (CHAN, W. C & WHITE, P. D. “Fmoc solid-phase peptide synthesis. A practical approch.” Oxford University Press; 2000).
In the end of the synthesis process the peptides were cleaved from resins by using the following cleavage solution: TFA:TES:H2O (95:2.5:2.5 v:v:v). After the cleavage reactions were completed, TFA was removed by bubbling N2 into the reaction tubes. Then, the peptides were washed six times with cold diisopropylic ether which is responsible for the removal of exceeding protecting groups and carbo-cations sequestrants, in addition to causing the precipitation of the peptides. The peptides were then extracted from the resins by means of washings with water, followed by filtration through a porous plate. This procedure was immediately followed by lyophilization of the resulting material.
After being lyophilized the peptides were purified by high performance liquid chromatography (HPLC) in a Äkta Explorer system, in a reverse phase column (C-18 or other). The peptides thus obtained were analyzed by mass spectrometry for quality control.
As example of a chemical modification of the synthetic peptides, the amino-terminal and carboxi-terminal of TsHpt17-25 (SEQ ID NO:1) were modified. The amino-terminal was acetylated using an acetic anhydride: diclorometane solution (1:1, v/v). The carboxi-terminal of TsHpt17-25 (SEQ ID NO:1) was amidated using the Rink Amide resin to synthesized this peptide, since after the cleavage of the peptide-resin bond, this resin causes the amidation of the carboxi-terminal of the peptide.
Surgical Procedures
Rats were subjected to a surgery for implanting polyethylene cannules into femoral vessels 24 hours before the experiments. The cannules were prepared from polyethylene with two different diameters (PE50 and PE10), fused by heating and filled with a solution of NaCl 0.9% w/v. The cannule of the femoral artery was intended to register the mean arterial pressure (MAP) and the one introduced in the femoral vein was used for administration of the peptides to be tested. The cannules were exteriorized in the interscapular of the animals.
Experiments for Potentialization of Hypotensive Effect of BK (
The peptides were diluted from a stock solution (1 mg/mL) with a solution of NaCl 0.9% w/v for injection at 10 μg/animal body (approximately 38 μg/Kg of animal body) resulting in a final volume of 200 μL of solution.
Firstly 1 μg of bradykinin (BK) was injected in a final volume of 100 μL. Later, 2 μg of bradykinin were injected with the same final volume, and the ΔMAP was calculated again. The peptides were, then, administrated intravenously and the MAP variation was measured.
MAP variation (ΔMAP) was calculated by means of the following equation:
ΔMAP=MAPfinal−MAPinitial
After administration of the peptide, 1 μg of BK was injected again in intervals of 10 minutes for 120 minutes and the ΔMAP was calculated to evaluate whether that variation is near a BK control (1 or 2 μg).
As an example, the scorpion's native peptide TsHpt-I was injected in bolus (35 μg/Kg), single dose, in normotensive male Wistar rats—weighting 250 to 300 g. After 40 minutes of the intravenous administration (i.v.), the peptide was able to double the effect of a single dose of BK (1 μg). This effect was observed up to 2 hours after the administration. A rapid and strong hypotensive response was observed after the injection of TsHpt-I (
Experiments for Evaluation of Hypotensive Effect of Synthetic Peptides in Hypertensive Rats (SHR and TGR) (
As a control condition MAP and heart rate of animals were observed and noted at intervals of 2 minutes during 1 hour. In the event MAP figures were lower than 150 mmHg, the animal was not considered as hypertensive and the experiment was interrupted. If MAP values were equal of higher than 150 mmHg, the peptides were administrated intravenously (100 μg/rat) and the MAP profile and heart rate (HR) of the animals were monitored for 6 hours with measurements of MAP and HR at each 2 minutes.
As example, the peptide TsHpt17-25 (SEQ ID NO:1) (303 μg/Kg) was intravenous injected in SHR hypertensive rats.
Evaluation of Vasodilating Effect in Rats Aorta Rings (
After being sacrificed, male Wistar rats had their thoracic aorta carefully removed, isolated from its fat and conjunctive tissue, and incubated with Krebs-Henseleit solution (mmol/L): NaCl 110.8; KCl 5.9; NaHCo3 25.0; MgSO4 1.07; CaCl2 2.49; KH2PO4 2.33; glucose 11.51. The aorta was, then, cut into rings (3-4 mm) that were connected to metallic rod coupled with isometric transducers. This preparation was kept in Krebs-Henseleit solution aired with carbogen (95% O2 and 5% CO2). Tension (1 g) was regulated during the first hour for stabilizing the preparation, the solution being changed at intervals of 15 minutes to avoid the accumulation of metabolism products. After the stabilization period, the vessels were pre-contracted with phenylephrine (0.1 μM) and, during the tonic contraction phase, cumulative concentration-answer curves with the synthetic peptides (10−10 at 3×10−7) were built.
As an example, endothelium-containing aortic rings pre-contracted with phenylephrine, the synthetic TsHpt-I (
Telemetry and Gavage in SHR Rats
A radio transducer was implanted in the stomach of hypertensive rats (SHR strain) for measuring, amongst other variables, the heart rate (HR) and mean arterial pressure (MAP).
The synthetic peptides were resuspended in saline solution (0.9% NaCl w/v) and orally administrated as a single dose (2.5 mg/KG; 0.1 mL per 100 g rat body weight). MAP and HR were measure by telemetry after the synthetic analog TsHpt21-25 (SEQ ID NO:5) was administrated by gavage. This analog was able to induce a reduction on MAP that was maintained regular up to 72 hours.
| Number | Date | Country | Kind |
|---|---|---|---|
| PI 0202157-9 | Jun 2002 | BR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10517097 | Jul 2005 | US |
| Child | 11687785 | Mar 2007 | US |
