DE10220188A1 - New antagonists of calcitonin gene-related peptide or amylin, useful for treating or preventing e.g. migraine or inflammation, are specific binding nucleic acids - Google Patents

Abstract

An antagonist (I) of CGRP (calcitonin gene-related peptide), especially a nucleic acid, preferably one that binds to CGRP, is new. Independent claims are also included for: (1) antagonist (Ia) of amylin that is a nucleic acid, preferably one that binds to amylin; (2) antagonist (Ib) of the CGRP or amylin receptors, preferably one that binds to a ligand of the receptor, particularly to CGRP or amylin; (3) nucleic acid (II) that binds to CGRP, amylin or an amyloid polypeptide; (4) complex of CGRP or amylin with a (I); (5) methods of screening for CGRP and amylin antagonists or agonists; and (6) kits, for detecting CGRP or amylin, containing (II).

Description

The present invention relates to antagonists of CGRP, antagonists of CGRP1- Receptor, CGRP binding nucleic acids, the use of such nucleic acids as Antagonists of CGRP and / or the CGRP1 receptor system, the use of one of the mentioned nucleic acids for the manufacture of a medicament, a composition, in particular pharmaceutical composition comprising said nucleic acid (s), a Complex comprising CGRP and one of said nucleic acids, the use of the said nucleic acids for the detection of CGRP and a method for screening CGRP antagonists and a kit for the detection of CGRP.

Hippocrates already described the visual symptoms of migraines, and also those of Egypt These headaches were not unknown to medicine, as papyrus finds testify. While In the 17th century it was recognized that the vessels of the body played an important role in Play migraine headache. It was already known back then that migraines, in addition to the seriousness the attacks, benign and hereditary, and of the seasons, of air pressure and of Consumption of certain foods depends (Willis, T., Cerebri anatome, Martin Allestry, London, 1664). After Willis (Willis, T., Cerebri anatome, Martin Allestry, London, 1664) headache is caused by a slowly developing vasospasm that occurs in the Periphery of the vascular system begins to fire. Another important aspect of migraine Pathophysiology emerged in the 19th century. Liveing (Liveing, E., Churchill, London, 1873, 1-512) attributed migraines to brain dysfunction caused by "nerve storms" arises within the brain. He believed that there was a relationship between migraines and Epilepsy exists because both are caused by central nervous system discharges. The Studies by Ray and Wolff (Ray, B.S. et al., 1940, Arch. Surg., 41, 813-856) demonstrate that only the large cerebral arteries of the skull base and the meningeal (dural) Arteries and veins are sensitive to harmful stimuli. That drew interest to them intercranial vessel walls as putative source of pain for headache.

According to the 1998 IHS classification, there are two main groups of migraines: Migraines with and migraines without aura. The earlier division into simple, classic and complicated migraines were given up.

Migraines are now understood as a neurovascular dysfunction, of which about 10% the adult population are affected. This disease is characterized by Attacks of intense recurrent headaches (Doods, H., 2001, Current opinion in investigational Drugs 2 (9), 1261-1268), nausea and exaggerated sensitivity to external stimuli such as light and noise. The often half-sided headache (Hemikranie) are pulsating and throbbing and usually occur on one side. Frequently the side of the headache changes from one migraine attack to another or even during an attack. The attack is often accompanied by other symptoms. The Those affected complain of anorexia, nausea and vomiting. They are particularly light and sensitive to noise and react extremely sensitively to smells and show nervous and Vision problems. The headache disappears between the migraine attacks. Before and After the attacks mood and appetite, the fluid balance and the Change bowel function.

The pathophysiology of migraines is still under discussion. The predominant current opinion is that migraine is a neurovascular disease whose The trigger mechanism may be localized in the CNS. This triggering leads ultimately for vasodilation with subsequent activation of trigeminal afferent senoric neurons and central "nociceptive" neurons in higher pain centers (Hargreaves, R.J. et al., 1999, Can. J. Neurol. Sci. 26, 512-519). There is some evidence and Evidence confirming the involvement of CGRP in migraines. CGRP is abundant in trigeminal sensory nerves and one of the most potent known vasodilators. Of another is the CGRP1 receptor on endothelial cells of the meningeal arteries expressed. CGRP is believed to play a key role as a mediator of the meningeal vasodilation. In addition to these physiological considerations, there are some animal studies that support involvement of the CGRP1 receptor in migraines. The Stimulation of the trigeminal ganglion in anesthetized rats leads to meningeal Vasodilation, which can be inhibited by the αCGRP antagonist CGRP8-37 (Kurosawa, M. et al., 1995, Br. J. Pharmacol. 114, 1397-1402). This experiment implies an increased CGRP level after trigeminal stimulation, which was already described by Goadsby in 1993 and Edvinsson (Goadsby, P. et al., 1993, Ann. Neurol., 33, 48-56).

Analysis of CGRP plasma concentrations in human subjects showed that the Concentration of CGRP in plasma is an important parameter in migraine diagnosis. It were elevated plasma concentrations in patients with acute migraines, in patients with Cluster headache and in people after trigeminal Stimulation (Edvinsson, L. et al., 1994, Cephalalgia 14, 320-327) was found. In Consistent with the animal studies just described, the increased CGRP- Concentrations described in migraine sufferers by sumatriptan or Dihydroergotamine can be reduced (Goadsby, P. et al., 1993, Ann. Neurol. 33, 48-56).

As part of the therapy of migraines, especially acute migraines and before training the Full screen of migraine headache is still considered the drug of choice of 1-1.5 g of acetylsalicylic acid (e.g. aspirin). In addition to acetylsalicylic acid 1 to 1.5 g of paracetamol are given. Alternatives to acetylsalicylic acid are NSAIDs d. H. non-steroidal anti-inflammatory agents, and non-steroidal anti-inflammatory drugs, such as As naproxen, diclofenac or ibuprofen.

The so-called triptans have proven particularly effective in the treatment of migraines proven to be currently the most potent therapeutic agent for acute moderate treatment to severe migraine attacks (Tepper, S.J. et al., 1999, CNS Drugs 12, 403-417; Doods, H., 2001, Current opinion in investigational Drugs 2 (9), 1261-1268).

Because of their high specificity for 5HT _{1B / D} receptors, they enable a very effective and safe treatment of acute migraine attacks, even with long-term use, taking into account the contraindications. Looking at the risks and secondary costs from the z. T. serious ergotamine side effects or insufficiently treated migraine attacks, the use of these currently most expensive, but also the most potent migraine therapeutic agents seems justified in many cases. In the trigeminovascular system, which is so important for migraines, the triptans also inhibit the release of vasoactive and algogenic neuropeptides (CGRP, neurokinins) from nociceptive trigeminal nerve endings and thereby prevent the initiation or maintenance of the neurogenic inflammation perivascularly, in particular the dura arterial site, the likely origin of migraine headache. After the successful introduction of sumatriptan, some new triptans were developed and approved on the market. These differ mainly in the beginning of the effect and the oral bioavailability. Although the triptans are effective and well tolerated, there are limitations for this class of substance. The occurrence of chest pressure, tightness / tension and anxiety often indicate angina pectoris in up to 15% of patients (Doods, H., 2001, Current opinion in investigational Drugs 2 (9), 1261-1268; Brown, EG et al Eur. Neurol., 31, 339-344). The use of sumatriptan has also been associated with myocardial infarction (Ottervanger, JP et al., 1993, Lancet, 341, 861-862) and cardiac arrest in cardiovascular risk patients (Kelly, KM, 1995, Neurology, 45, 1211-1213). It was also observed that the administration of sumatriptan, rizatriptan (Merck & Co. Inc.) and zolmitriptan (Glaxo Wellcome plc / AstraZeneca plc) induced a slight increase in blood pressure (De Hoon, JNJM et al., 2000, Clin. Pharmacol. Ther. , 68, 418-426). The vasoconstrictive activity of the triptans on the coronary system has been clearly demonstrated in vitro and in vivo, although it appears that the triptans act selectively on cerebral vessels (Doods, H., 2001, Current opinion in investigational Drugs 2 (9), from 1261 to 1268).

Another group of active ingredients to be used are the ergotamines.

The release of CGRP in primary headache, the pharmacology of the trigeminovascular system, the concept of neurogenic inflammation (Moskowitz, M. A. et al., 1993, Brain Metab. Rev. 5, 159-177) and the response to triptans (Humphrey, P.P.A. et al., 1991, Trends Pharmacol. Sci., 12, 444-446) are key elements in the pathology of the Migraines (Edvinsson, L., 2001, Pharmacology & Toxicology 89, 65-73). Accordingly, it has the latest pharmacological research has focused on this neuropeptide.

The 37 amino acid neuropeptide αCGRP, calcitonin gene-related peptide, was identified in 1982 as an extremely potent vasodilator (Amara et al., 1982, Nature 298, 240-244). CGRP arises from alternative splicing of the CGRP gene. In addition to the αCGRP, there is a second CGRP, βCGRP, which has a high sequence homology to the former, but is transcribed by another gene. Both peptides show similar biological effects like vasodilation, increased blood pressure, hypotension and tachycardia. αCGRP and calcitonin result from alternative splicing of the calcitonin gene (Amara, GS et al., 1982, Nature 298, 240-244). The structure for hCGRP was determined in part by ¹ H NMR. The peptide consists of a defined N-terminal loop, which is formed from amino acids 2 to 7 by linking two cysteines via a disulfide bridge, which is followed by about three turns of an α-helix. Towards the C-terminus there follows a poorly defined kink, which in turn leads to an unstable structure at the C-terminus itself (Breeze, AL et al., 1991, Biochemistry, 30, 575-582). Furthermore, the C-terminal phenylalanine is in amidated form.

Ergotamine supplements are classic drugs used to stop a migraine attack due to the possible side effects are not entirely unproblematic. The danger of one Getting used and triggering an additional permanent headache increases with increasing Dosing frequency. For this reason, should not exceed 6 mg per week Ergotamine tartrate and no more than 4 mg per migraine attack. In principle, the same applies here that in the case of migraine headache, the use of Mixed preparations (e.g. ergotamine tartrate with caffeine or prophyphenazone, codeine, Paracetamol etc.) should be strictly avoided. At this therapy level, 1-1.5 mg Dihydroergotamine (Hydergin®) i. m. or very slowly i. v. be tried. Especially at pronounced vegetative migraine side effects has the additional dose of 1-2 mg Flunitrazepam (Rohypnol®, a sleep aid) has proven itself. Especially under that Aspect of saving painkillers, especially since the patients in this situation anyway Need to lie down. Are the migraine headaches from nausea and Accompanied vomiting (possibly even before the expected onset of these symptoms) is Administration of metoclopramide (Paspertin®) very effective. It is beneficial to do this Take substance before an analgesic because metoclopramide stops bowel activity increases and thus promotes the absorption of other administered substances. Alternatively, you can the dopamine antagonist domperidone (Motilium®) can also be used.

A number of CGRP antagonists as well as those derived therefrom are in the prior art Derivatives known. For example, a Quinine analogue is a CGRP antagonist in the international patent application with publication number WO 97/09046 described. However, these compounds show only a weak affinity for the human CGRP receptor in the micromolar range and are therefore not of great importance.

A first potent non-peptide CGRP receptor antagonist is the compound BIBN-4096BS, as described in DE 199 11 039. This substance is a Lys-Tyr dipeptide derivative and has a high affinity for the human CGRP1 receptor (K _i = 14 , 4 pm). In studies on cerebral vessels, BIBN-4096BS was able to reverse the CGRP-mediated vasodilation (Doods, H. et al., 2000, Br. J. Pharmacol., 129, 420-423). Although high doses of CGRP are cardioprotective, the α-CGRP antagonist BIBN-4096BS had no negative effects on myocardial infarction or the release of creatine phosphate kinase.

A cyclopropyl derivative was developed based on the structure of BIBN-4096BS which the dipeptide core was replaced by a cyclopropyl ring. This connection is Subject of the international patent application with the publication number WO 01/32648. Other CGRP receptor antagonists are the subject of international Patent application with publication number WO 01/32649, the naphthalenes, Describe piperidines, imidazoles and quinazolines as CGRP receptor antagonists.

Other structural classes that are the subject of investigations, they as CGRP receptor Antagonists to use are 3,4-dinitrobenzamines, such as in the international patent application with publication number WO 98/09630 described, as well as 4-sulfinylbenzanilides, as in the international patent application with the Publication number WO 98/56779. Show detailed attachment analysis however, that these substances have irreversible binding properties. Furthermore show restrictions, especially some representatives of 4-sulfinylbenzanilides in such a way that they have a comparatively low solubility and oral availability as well have a short half-life of approx. 10 minutes, which is an intensive in vivo Excludes characterization.

The present invention was therefore based on the object of providing a means which is used to treat migraines and the related Form of other diseases is suitable. Another of the present invention underlying task is to provide an antagonist for CGRP as well as the CGRP1 receptor system. Finally, it is an object of the present invention to provide a To provide methods with the CGRP antagonist, in particular in the context of a Screening method can be provided.

According to the invention, the problem is solved in a first aspect by an antagonist of CGRP, where the antagonist is a nucleic acid and preferably the Nucleic acid binds to CGRP.

In one embodiment it is provided that the CGRP is α-CGRP.

In an alternative embodiment it is provided that the CGRP is β-CGRP.

In a second aspect, the object is achieved according to the invention by an antagonist of the CGRP1 receptor, wherein the antagonist is a nucleic acid and wherein preferably the nucleic acid binds to a ligand of the receptor and wherein more preferably the ligand is CGRP.

In one embodiment it is provided that the ligand is α-CGRP.

In an alternative embodiment it is provided that the ligand is β-CGRP.

In a further embodiment it is provided that the nucleic acid has at least one L- Includes nucleotide.

In a preferred embodiment it is provided that the antagonist is an L- Is nucleic acid.

In a third aspect, the object is achieved according to the invention by a nucleic acid, that binds to CGRP.

In one embodiment it is provided that the CGRP is α-CGRP.

In an alternative embodiment it is provided that the CGRP is β-CGRP.

In a fourth aspect, the object is achieved according to the invention by a nucleic acid with a sequence, the sequence being selected from the group comprising the Sequences according to SEQ ID No. 1 to SEQ ID No. 244th

In one embodiment of the third and fourth aspects it is provided that the Nucleic acid comprises at least one L nucleotide.

In one embodiment of the third and fourth aspects it is provided that the Nucleic acid is an L-nucleic acid.

In a further embodiment of the third and fourth aspects it is provided that the Nucleic acid is selected from the group comprising DNA, RNA and combinations from that.

In yet another embodiment of the third and fourth aspects it is provided that the affinity of the nucleic acid is less than 0.5 µM, preferably less than 0.1 µM, is preferably less than 0.05 µM and most preferably less than 0.01 µM.

In yet another embodiment of the third and fourth aspects, that the affinity of the nucleic acid is more than 250 nM, preferably more than 100 nM, more preferably is more than 50 nM and most preferably more than 10 nM.

In one embodiment of the third and fourth aspects it is provided that the Nucleic acid includes a minimal binding motif.

In a further embodiment of the third and fourth aspects it is provided that has a length, the length being selected from the group consisting of lengths from 15 to 150 nucleotides, 20 to 120 nucleotides, 30 to 120 nucleotides, 40 to 100 nucleotides, 40 to 70 nucleotides and 50 to 70 nucleotides, 30 to 50 and most preferably 25 to 45 Includes nucleotides.

In yet another embodiment of the third and fourth aspects it is provided that the nucleic acid has a two, three or more part structure.

In a fifth aspect, the object is achieved according to the invention through the use one of the nucleic acids according to the invention as an antagonist of CGRP and / or the CGRP1 receptor system.

In a sixth aspect, the object is achieved according to the invention through the use one of the nucleic acids according to the invention for the manufacture of a medicament.

In a seventh aspect, the object is achieved according to the invention through the use of an antagonist according to the invention for the manufacture of a medicament.

In one embodiment the uses according to the two above aspects provided that the medication is for the treatment of a disease arising from the Group selected is the migraine, cluster headache, loss of appetite, nausea, Vomiting, neurogenic inflammation, especially neurogenic inflammation, with others Is mediated neuropeptides, vasodilation, increased blood pressure, hypotension, tachicadia, Diseases related to activation of trigeminal afferent sensory neurons and central "nociceptive" neurons, especially higher pain centers, decrease, and chronic inflammatory pain, includes and / or for the treatment of pain, especially chronic pain, acute pain, inflammatory pain, more visceral Pain and neuropathic pain.

In an eighth aspect, the object is achieved according to the invention by a Composition comprising an inventive and preferably a pharmaceutically acceptable carrier.

In a ninth aspect, the object is achieved according to the invention by composition comprising an antagonist according to the invention and preferably one pharmaceutically acceptable carrier.

In a tenth aspect, the object is achieved according to the invention by complex comprising CGRP and a nucleic acid according to the invention.

In an eleventh aspect, the object is achieved according to the invention through the use a nucleic acid according to the invention for the detection of CGRP, preferably α- CGRP or β-CGRP and most preferably human α-CGRP or β-CGRP.

• - Bereitstellen eines Kandidaten-CGRP-Antagonisten,
• - Bereitstellen einer erfindungsgemäßen Nukleinsäuren,
• - Vorsehen eines Testsystems, welches ein Signal in Gegenwart eines CGRP- Antagonisten ergibt, und
• - Bestimmen, ob der Kandidaten-CGRP-Antagonist ein CGRP-Antagonist ist.

In a twelfth aspect, the object is achieved according to the invention by methods for screening CGRP antagonists comprising the following steps:

• Providing a candidate CGRP antagonist,
• Provision of a nucleic acid according to the invention,
• Provision of a test system which gives a signal in the presence of a CGRP antagonist, and
• - Determine whether the candidate CGRP antagonist is a CGRP antagonist.

In one embodiment it is provided that the CGRP is α-CGRP and / or β-CGRP, preferably human α-CGRP and / or β-CGRP.

• - Bereitstellen von CGRP, bevorzugt immobilisiertem CGRP,
• - Bereitstellen einer erfindungsgemäßen Nukleinsäuren, bevorzugterweise einer markierten erfindungsgemäßen Nukleinsäure,
• - Zugabe eines Kandidaten-CGRP-Agonsiten, und
• - Bestimmen, ob der Kandidaten-CGRP-Agonist ein CGRP-Agonist ist.

In a thirteenth aspect, the object is achieved according to the invention by methods for screening CGRP agonists comprising the following steps:

• Provision of CGRP, preferably immobilized CGRP,
• Provision of a nucleic acid according to the invention, preferably a labeled nucleic acid according to the invention,
• - addition of a candidate CGRP agonsite, and
• - Determine whether the candidate CGRP agonist is a CGRP agonist.

In one embodiment it is provided that the determination takes place in that it is determined whether the nucleic acid is displaced by the candidate CGRP agonist.

In a fourteenth aspect, the object is achieved according to the invention by a kit for the detection of CGRP, preferably α-CGRP or β-CGRP, comprising one nucleic acid according to the invention.

The present invention is based on the surprising finding that it is possible to generate nucleic acids specifically binding to CGRP. After extensive evidence for the existence of CGRP in pain and the development of migraines in is involved in their various forms of training, it follows that such Nucleic acids used as antagonists for CGRP or the CGRP1 receptor system can and in so far as active pharmaceutical ingredients in the treatment of Diseases of the type of migraine. It is particularly noteworthy that it CGRP is a comparatively small peptide against which binding Nucleic acids are difficult to generate. The amino acid sequence of human αCGRP and βCGRP differ in three amino acids, the amino acid sequence of αCGRP and βCGRP and the rat differ in an amino acid. The various sequences are described in Hakala and Vihinen (Hakala J. M. L. and Vihinen M., 1994, Protein Engineering 7 (9), 1069-1075. (Accession numbers: human αCGRP P06881, human βCGRP P10092, advice αCGRP P01256, advice βCGRP P10093.

This application is essentially based on the observation that CGRP in neural tissue and especially in somatic sensory cells is abundant and often co-expressed with other neuropeptides such as substance P, and the further results of pain and migraine research described below.

Immunocytochemical studies show that substance P is almost always small in size with CGRP DRG neurons (dorsal root ganglion) are associated, whereas CGRP is also without substance P is observed (Wiesenfeld-Hallin, Z. et al., 1984, Neurosci. Lett. 52, 199-203). The Release of CGRP in the periphery leads to vasodilation and together with other neuropeptides, such as substance P, for neurogenic inflammation. CGRP is also in the Dorsal horn of the spinal cord in response to harmful stimulations in the periphery released and thus provides an application and investigation site for the application of the nucleic acids, antagonists and agents according to the invention.

The CGRP1 antagonist CGRP8-37 has been tested in different pain models. To antagonize endogenous CGRP, an anti-CGRP antiserum has been used in various Pain models tested after intrathecal administration. The generation of αCGRP-deficient Mice and behavioral experiments close the picture. The anti-nociceptive effect of CGRP8-37 could be used in various pain models such as phenylquinone-induced (PQ) Writhing (Saxen, M.A. et al., 1994, Life Sciences 55, 1665-1674), acetic acid-induced Writhing (Friese, N. et al., 1997, Regulatory Peptides 70, 1-7), visceral pain (colorectal distension model, Plourde, V. et al., 1997, Am. J. Physiol, 35, G191-G196), Burn pain (heat-induced hyperalgesia, Lofgren, O. et al., 1997, Neuropeptides 31, 601-607), and neuropathic pain (spinal hemisection, Bennett, A.D. et al., 2000, Pain 86, 163-175). In mouse tail-flick, a model for acute pain, there was no effect to be watched.

In order to block the effect of the spinally released CGRP, an antiserum in tested various models for chronic pain. In chronic inflammatory Pain models such as adjuvant-induced arthritis (Kuraishi, Y. et al., 1988, Neurosci. Lett. 92, 325-329) or carrageenin-induced hyperalgesia (Kawamura, M. et al., 1989, Brain Res. 497, 199-203) showed the anti-CGRP antiserum anti-nociceptive activity. This antiserum can also cause repeated stress-induced hyperalgesia in rats prevent (Satoh, M. et al., 1992, Pain 49, 273-278).

The anti-nociceptive effects observed with both the truncated peptide CGRP8-37 and with the antiserum match well with the hyperalgesia observed in αCGRP-deficient knockout mice (Salmon, A.-M. et al., 1999, Neuroreport 10, 849-954). CGRP ^{- / -} mice show reduced hyperalgesia compared to the CGRP ^{+ / +} mice with chronic inflammatory pain that was triggered by formalin or capsaicin injections into the hind paw (Salmon, A.-M. et al., 2001, Nature 56, 357-358). A second αCGRP-deficient mouse was created to study the role of calcitonin. The CGRP ^{- / -} mice are born normally, are fertile and live a normal life. In a chronic arthritis model (kaolin-carragenin mix was injected into the knee joint), these mice do not develop secondary hyperalgesia compared to the wild type (Zhang, L. et al., 2001, Pain 89, 265-273).

As a result of these results, α-CGRP antagonists such as the CGRP- Antagonists or the nucleic acids according to the invention are also effective for the treatment of chronic inflammatory and visceral pain.

The nucleic acids according to the present invention are also intended to be such nucleic acids which are substantially homologous to the sequences specifically disclosed herein. The term essentially homologous should be understood herein to mean homology at least 75%, preferably 85%, more preferably 90% and most preferred is more than 95, 96, 97, 98 or 99%.

The term nucleic acid according to the invention or nucleic acid according to the present The invention is also intended to encompass those nucleic acids that are part of those disclosed herein Include nucleic acid sequences or nucleic acids, preferably to the extent include that said parts are involved in the binding of CGRP. This kind of Nucleic acids can be derived from those disclosed herein, for example by Shortening or truncation. The shortening is meant to be either on one or both ends of the nucleic acids disclosed herein. The shortening can also affect one Refer to nucleotide sequence within the respective nucleic acid or nucleic acid sequence, d. H. it can refer to one or more nucleotides between the 5'- and the 3'- terminal nucleotide. In connection with this, the deletion is supposed to shorten by so little such as a single nucleotide from the sequence of the nucleic acids disclosed herein include. Shortening can also cover more than one area of the invention Affect nucleic acid (s). Examples of the shortening of the nucleic acids are in the Sample part of the present description taught.

The nucleic acids according to the present invention can either be D-nucleic acids or L-nucleic acids. The nucleic acids are preferably L-nucleic acids. In addition, it is possible that one or more parts of the nucleic acid as D-nucleic acid (s) are formed, or at least one or more parts of the nucleic acid as L-nucleic acid are trained. The term "part" of the nucleic acid is meant to be as little as a nucleotide describe. Such nucleic acids are generally referred to herein as D- or L- Designated nucleic acid.

It is also within the scope of the present invention that the inventive Nucleic acids are part of a longer nucleic acid, this longer nucleic acid comprises several parts, at least one part of a nucleic acid according to the present Invention or part of it. The other part or parts of this longer one Nucleic acids can be either a D-nucleic acid or an L-nucleic acid. A any combination can be used in connection with the present invention. This other part or these other parts of the longer nucleic acid can have a function which is different from binding and in particular binding to CGRP. A possible function is to interact with other molecules, e.g. B. too To allow purposes of immobilization, cross-linking, detection or amplification.

The L-nucleic acids are therefore nucleic acids which consist of L-nucleotides preferably consist entirely of L nucleotides.

D-nucleic acids are thus those nucleic acids which consist of D-nucleotides preferably consist entirely of D nucleotides.

Regardless of whether the nucleic acid of the invention from D nucleotides, L- Nucleotides or a combination of both, the combination z. Legs random combination or a defined sequence of sequences of nucleotides that consists of at least one L-nucleotide or one D-nucleotide, the nucleic acid from one or more deoxyribonucleotide (s), ribonucleotide (s) or combinations of which exist.

The formation of the nucleic acids according to the invention as L-nucleic acid is one of a number advantages associated with reasons. The L-nucleic acids are enantiomers of the naturally occurring nucleic acids. However, the D nucleic acids are in aqueous Solutions and especially not in biological systems or biological samples stable due to the widespread use of nucleases. Naturally occurring nucleases, in particular nucleases of animal cells or tissues or cell or Body fluids are unable to break down L-nucleic acids. As a result the biological half-life of L-nucleic acid in such systems, including the human and animal body, significantly extended. Due to the lack of one Degradability of L-nucleic acids does not produce nuclease degradation products and it does to this extent no side effects due to this are observed. That aspect distinguishes the L-nucleic acid from all those other compounds used in therapy of diseases that rely on the presence of CGRP.

It is also within the scope of the present invention that the inventive Nucleic acids, regardless of whether they are D-nucleic acids, L-nucleic acids or D, L- Nucleic acids are present, or whether they are present as DNA or RNA, as single-stranded or double-stranded nucleic acids can be present. Typically they are nucleic acids according to the invention single-stranded nucleic acids which have a defined Having a secondary structure as a result of the primary sequence and also forming tertiary structures can. However, the nucleic acids according to the invention can also be double-stranded in in the sense that there are two mutually complementary strands due to hybridization are paired with each other. This gives the nucleic acid a stability that is beneficial is when the nucleic acid in the naturally occurring D-form instead of the L- Form is present.

The nucleic acids according to the invention can also be modified. A special one advantageous modification in connection with the present invention represents the Design of the nucleic acid (s) according to the invention in which at least one, preferably several, and most preferably all, forming the nucleic acid Pyrimidine nucleotides at the 2 'position of the ribose part of the respective nucleotides a 2- Have fluorine group.

Further examples of a modification of the nucleic acids according to the invention can be such modifications which relate to a single nucleotide of the nucleic acid and in State of the art are very well known. Relevant examples are, among others, described in Kusser, W. (2000) J Biotechnol 74, 27-38; Aurup, H. et al., 1994, Nucleic Acids Res 22, 20-4; Cummins, L.L. et al. 1995, Nucleic Acids Res 23, 2019-24; Eaton, B.E. et al., 1995, Chem Biol 2, 633-8; Green, L.S. et al., 1995, Chem Biol 2, 683-95; Kawasaki, A.M. et al., 1993, J Med Chem 36, 831-41; Lesnik, E.A. et al., 1993, Biochemistry 32, 7832-8; Miller, L.E. et al., 1993, J Physiol 469, 213-43.

The nucleic acids according to the present invention can be in one, two, three or be formed in several parts. Of the multi-part form is particularly the two-part or bipartite form preferred. A multi-part form of a nucleic acid according to the invention herein is in particular one that consists of at least two nucleic acid strands. These two nucleic acid strands form a functional unit, the functional one Unit is a ligand or binding molecule for a target molecule. The at least two Strands can be derived from one of the nucleic acids according to the invention by either cleaving an inventive binding to the target molecule Nucleic acid to form two or more strands, or by synthesis of one Nucleic acid, which is a first part of the complete nucleic acid according to the invention corresponds and a further nucleic acid which is a second part of the invention corresponds to complete nucleic acid. It is recognized that both the split and the synthesis can be used to produce a multi-part nucleic acid derived from more than the two strands described above by way of example. For the rest is regarding the multi-part forms of the nucleic acid according to the invention, that at least two strands of nucleic acid are typically different from two strands that are complementary to each other and hybridize with each other, although to some extent Complementarity between different nucleic acids (parts) can exist.

The nucleic acids according to the present invention typically have a high one Affinity for the target molecule. One way of affinity, expressed as Binding constant to determine the nucleic acids according to the invention is Use of the so-called biacore device known to those skilled in the art and is described, for example, in Jonsson, U. et al., 1991, Biotechniques, 11 (5), 620. Die Affinities were also measured by isothermal titration calorimetry (ITC) as in the Examples and in Haq, I. & Ladburg, J., 2000, J. Mol. Recognit. 13 (4): 188. As such, the affinity values described herein are as isothermal To understand titration calorimetry, taking the temperature for each Measurement was 25 ° C, unless otherwise stated. Another method used is applied manually; it is a so-called bead Assay. Here, constant concentrations of radioactively labeled nucleic acid are included different concentration of biotinylated target molecule such as a target Peptide put together. After a defined time, the complexes formed are transferred to the Addition of beads loaded with streptavidin removed from the solution and the respective The amount of radioactivity is determined. From the values obtained, the corresponding plot in graphs determines the binding constants. This bead Assay is more detailed and a. described in Example 3. Insofar as this refers to a In principle, all nucleic acids according to the invention are referred to understood nucleic acids according to the invention or all nucleic acids disclosed herein unless otherwise stated.

It is also within the scope of the present invention that the sequences according to the invention either entirely or in part from the randomized portion of the members of a Nucleic acid library originate as the starting material for the selection process be used. However, it is also within the scope of the present invention that the sequences according to the invention either completely or partially from the non- randomized part of the members of the nucleic acid library that is used as a starting material for serves the selection process, originate. Such is a non-randomized part for example the part used as the binding site for the amplification primer.

The nucleic acids according to the invention can be used for the generation or Manufacture of a drug. Such a medicament contains at least one of the nucleic acids according to the invention, optionally together with other pharmaceutically active Compounds, the nucleic acid (s) according to the invention preferably itself as pharmaceutically active compound acts. For example, one of the Nucleic acids according to the invention can be combined with a further active ingredient which Concentration influenced by CGRP. Generally, the combination of the The nucleic acids according to the invention with the further active ingredient are particularly advantageous to be when both components of CGRP and its release via different Attack mechanisms of action. A combination that is advantageous in this sense could for example from one of the nucleic acids according to the invention and a triptan, such as For example, sumatriptan exist because the two substances are different Address mechanisms of action. In the event of a migraine attack, you could get a combination Present the preparation and its effect as follows: CGRP occurring in plasma captured by a nucleic acid according to the invention and at the same time by Triptans reduce / prevent CGRP release. Such drugs include in preferred embodiments at least one pharmaceutically acceptable carrier. Such carriers can e.g. B. water, buffer, starch, sugar, gelatin and the like. Such carriers are known to those skilled in the art.

The diseases for which the nucleic acids according to the invention and those under it Use identified CGRP antagonists can be used in essential ones that belong to the form of migraines include migraines and without aura, simple migraines, classic migraines and complicated migraines. To include headache, especially recurrent headache, Nausea, vomiting, exaggerated sensitivity to external stimuli such as light and Noises, loss of appetite and disturbances in the fluid balance. The headache is especially one that appears to the patient as pulsating and throbbing. Typically, this headache occurs unilaterally. Other diseases listed under Use of the nucleic acids according to the invention and on the basis thereof Use identified candidate CGRP antagonists can be identified are vasodilation, increased blood pressure, hypotension and tachycardia, especially those Forms of the above disorders associated with migraines, especially a migraine attack. Other diseases listed under Use of the nucleic acid according to the invention or using the candidate CGRP antagonists identified according to the method of the invention are those diseases that are associated with activation of trigeminal afferent sensory neurons, with the activation of central nociceptive neurons and Combinations of activation of both classes of neurons go hand in hand or are connected. In particular, the neurons are the higher pain centers assigned. Other diseases in the aforementioned sense are in addition Acute pain is a condition associated with chronic inflammatory pain are. Such chronic inflammatory pain includes, in particular, that of CGRP can be caused along with other neuropeptides, such as substance P.

The nucleic acids according to the invention can also be used as a starting material for the design of pharmaceutical active substances (English drug design) are used. Basically there there are two possible approaches to this. One approach is to screen libraries from Compounds, such libraries of compounds preferably Libraries of low-molecular compounds (English low or small molecules) are. Such libraries are known to those skilled in the art. Alternatively, you can according to the present invention the nucleic acids for the rational design of Active ingredients are used.

The rational design of active ingredients can be the starting point of any of the Take nucleic acids according to the present invention and comprises a structure in particular a three-dimensional structure that is similar to the structure of the invention Nucleic acid (s) is or is identical to the part of the structure of the invention Nucleic acid (s) that mediate binding to CGRP. In any case, such shows Structure still the same or at least a similar binding behavior as that nucleic acid (s) according to the invention. In either a further step or as one alternative steps are preferred in the rational design of active ingredients three-dimensional structure of those parts of the nucleic acids binding to CGRP mimicking chemical groups, which are preferably different from nucleotides and Nucleic acids. Through this imitation, also known as mimicry, a connection can be established are constructed, which is different from the nucleic acid or the nucleic acids, which as Starting materials for the rational design of the active ingredient was used. A such a compound or active ingredient is preferably a small molecule molecule) or a peptide.

In the case of screening compound libraries, such as when using a Competitive tests that are known to those skilled in the art can be used to perform appropriate tests CGRP analogs, CGRP agonists or CGRP antagonists can be found. such competitive assays can be structured as follows. The nucleic acid according to the invention, preferably a Spiegelmer which forms L-nucleic acid binding as target molecule is coupled to a fixed phase. To identify CGRP analogs, use added neuropeptides labeled to the test system. A potential Analogue would compete with the CGRP molecules that bind to the Spiegelmer what with a decrease in the signal obtained from the corresponding marker would go hand in hand. Screening for agonists or antagonists can be used a cell culture test system known to those skilled in the art.

In a further aspect, the nucleic acids according to the invention can be used for the Target validation are used due to their characteristic binding behavior CGRP. The nucleic acids according to the invention can be used in an ex vivo organ model used to study the function of CGRP. There are basically two ex vivo models in which CGRP agonists / antagonists can be tested. in the Guinea pig atrium can be tested in the antagonists for the CGRP2 receptor Rats Vas deferens model may have specificity for the antagonist CGRP1 receptor to be checked.

In a further aspect of the invention, this relates to a complex comprising CGRP and at least one of the nucleic acids according to the invention. It is surprising it has been found that the nucleic acids according to the invention are relatively rigid and one adopt a precisely defined structure and in this respect also the target molecule, d. H. the CGRP itself, stamp a comparatively rigid structure, with the CGRP due to its length is generally understood to be comparatively flexible.

The kit according to the present invention may include at least one or more of the comprise nucleic acids according to the invention. In addition, the kit can have at least one or include multiple positive or negative controls. As positive controls z. B. CGRP against which the nucleic acid according to the invention has been selected, or to which binds them, preferably in liquid form. As a negative control, other a peptide can be used, which is different in terms of its biophysical Properties behaves similarly to CGRP, but not due to the invention Nucleic acids are recognized or a peptide with the same amino acid composition sequence different from CGRP.

The kit can further comprise one or more buffers. The different components can be in the kit in dry or lyophilized form, or dissolved in a liquid available. The kit can comprise one or more containers, which in turn contain one or more may contain several of the components of the kit. The vessels preferably contain Reaction approaches, such as those required for a single experiment Use of one or more components of the kit are required.

The present invention is further illustrated by the following figures and examples, from which further features, embodiments and advantages result. It shows

Fig. 1 shows the principle of an RNA selecting at CGRP binding nucleic acids comprising a connection step with the partial steps of denaturation and folding as well as connection-washing elution and amplification step comprising the step of reverse transcription-PCR and T7 transcription and purification;

Fig. 2 shows the course of a selection of 2'-fluoro-modified CGRP binding to nucleic acids, in particular

Fig. 2A, the CGRP concentration and the corresponding percentage binding of the F-RNA pool used in each round of selection, and

Figure 2B is a graphical representation of the values given in Figure 2A;

FIG. 3 shows the result of a sequence analysis of the obtained under Example 1 CGRP binding nucleic acids;

Fig. 4-11, the secondary structure of various binding to CGRP nucleic acids, such as those generated in the context of Example 2;

12 shows the course of the pre-columns during the selection of binding to CGRP nucleic acids.

Fig. 13 the course of the CGRP binding 2'-F-RNA and peptide concentration of selection approach NA using neutravidin as selection matrix;

Fig. 14 the course of the binding of CGRP 2'-F-RNA and peptide concentration of selection approach SA-1 and SA-0.1 using streptavidin magnetic beads derivatized as a selection matrix;

15 shows the reaction scheme for the synthesis of 5'-DMT-2'-fluoro-L-uridine phosphoramidite.

FIG. 16 is the view of 2'-fluoro-L-cytidine phosphoramidite from 2'-fluoro-L-uridine;

17 shows the synthesis of 2'-fluoro-L-cytidine from 2'-fluoro-L-uridine.

. Figure 18 shows the sequences of the output sequences and truncated sequences of the CGRP binding nucleic acids of Example 2;

FIG. 19 is the percent binding of various CGRP-detecting nucleic acids, as described in detail in Example 3; NA-A here means NeutrAvidin agarose with affinity elution, NA-D means NeutrAvidin agarose with denaturing elution, SA-1 means streptavidin beads with 1 nM peptide and SA 0.1 nM peptide concentration

FIG. 20 is the result of a competition analysis in accordance with Example 3 of selected different CGRP binding nucleic acids;

FIG. 21 is a representation of various experiments to determine the dissociation constants CGRP binding nucleic acids;

FIG. 22 is a further representation of experiments to determine the dissociation constants of various CGRP binding nucleic acids;

Fig. 23 shows the inhibition of cAMP production by human CGRP binding Spiegelmers;

Fig. 24 shows the inhibition of cAMP production by rat CGRP binding Spiegelmers,

Fig. 25 binding studies with the different purified spiegelmers 732_045 (55mer) and 732_096 (59mer) in the ^[35 S] GTPγS assay

Fig. 26-27, the secondary structure of various binding to CGRP nucleic acids, such as those generated in the context of Example 2, and which are composed of more than one nucleic acid strand,

Fig. 28, 30, 32, 34, 36 graphically the course of the different rounds of selection conditions,

Fig. 29, 31, 33, 35, 37 a table the course of the different rounds of selection conditions,

Fig. 38 depicts the dose response curve for CGRP,

Fig. 39 to 42 the course of calorimetric determination of the binding constants of various CGRP binding nucleic acids;

Fig. 43 to 47 show the sequences of different, binding in the context of Example 4 obtained CGRP nucleic acids, and

FIGS. 48 and 49 the sequences of different CGRP-binding nucleic acids obtained in the context of Example 3.

example 1 Production of 2'-fluoro-modified Spiegelmeres

Based on the method for identifying nucleic acids, in particular D-nucleic acids, which bind to a target molecule, as illustrated, for example, in FIG. 1, 2'-fluoro-modified Spiegelmers using 2'-fluorinated RNA molecules were used.

The fluoro-modified nucleic acids (F-RNA) used in the selection process differ from biologically occurring RNA by the presence of a Fluoro group on the 2'-carbon of the ribose. Here fluorine replaces the natural one Ribose hydroxyl group. This modification was here on pyrimidine nucleotides limited. Such artificial nucleic acids are created enzymatically with a mutant T7 polymerase (Epicenter, Madison, WI, USA), which is more tolerant to modified Is nucleotides using 2'-fluoro-UDP and 2'-fluoro-CTP. The mutation of the T7 polymerase enables the efficient incorporation of fluoro-modified ones Nucleoside triphosphates.

A. Materials

GTP, ATP, and dNTPs were from Larova, Berlin; Fluoro-dUTP and Fluoro-dCTP purchased from TriLink, San Diego, USA. The T7 DNA / RNA polymerase for the production of fluorinated RNA was obtained from Epicenter, USA. The enzymes Taq DNA Polymerase and Superscript II Reverse Transcriptase were from Life Technologies, the kit for the Reverse Transcriptase / Taq Polymerase was from Qiagen. Radioactive ³² [P] -α-GTP for labeling the F-RNA was obtained from Hartmann, Braunschweig.

D-CGRP was first developed by JERINI AG, Berlin, and later by Bachem, Heidelberg synthesized. The peptide used for selection carries a biotin group Carboxyl terminus to separate unbound nucleic acids using the biotin To enable streptavidin or biotin-neutrAvidin interaction. For that were NeutrAvidin agarose from Pierce and Streptavidin Paramagnetic Particles from Roche used. Unbiotinylated peptide (also from JERINI, Berlin, and Bachem, Bubendorf, Switzerland) was used in the first seven rounds for affinity elution.

DNA pool

The DNA for the start pool was synthesized in-house and is based on the described pool PB40 with the sequence 5'-GGA GCT CAG CCT TCA CTG CN ₄₀ -GGC ACC ACG GTC GGA TCC AC-3 '(Burgstaller & Famulok, 1994, Angew. Chem. Int. Ed. 33, 1084).

The 5 'and 3' primers had the sequence
PB40-R-For: 5'-TCT AAT ACG ACT CAC TAT AGG AGC TCA GCC TTC ACT GC-3 'and
PB40-R-Rev: 5'-GTG GAT CCG ACC GTG GTG CC-3 '.

The single-stranded DNA was checked for its amplifiability with radioactive primers; a complexity of 1.41 × 10 ¹⁴ molecules / nmol DNA was derived from this. The single-stranded DNA was processed by PCR in a total volume of 8 ml to double-stranded DNA.

B. Selection steps Denaturation and folding of the fluorinated RNA

All non-enzymatic steps of the selection with the exception of denaturation were carried out in selection buffer (HEPES-KOH, pH 7.5; 150 mM NaCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ and 0.1% Tween 20). The denaturation was carried out for 3 minutes at 94 ° C. in a selection buffer without Tween 20, MgCl ₂ and CaCl ₂ . After denaturation, the enzymatically produced F-RNA was first incubated in selection buffer for 30 minutes at 37 ° C., MgCl ₂ and CaCl ₂ were added and the folding was continued again at 37 ° C. for 30 minutes.

connection

Following the folding, the F-RNA was first incubated with the matrix (NeutrAvidin agarose or streptavidin paramagnetic particles) at 37 ° C. for 15 minutes without peptide. This so-called pre-selection served to pre-isolate potential matrix binders. After this incubation step, the F-RNA was separated from the matrix, mixed with the concentrations of biotinylated CGRP shown in FIG. 2A and left at 37 ° C. for at least 3 hours. The biotin-binding matrix was then added to the binding mixture and incubated again at 37 ° C. for 5-10 minutes. The matrix was separated from the solution and washed with selection buffer. The wash volume used in the first rounds was 5 to 10 times the amount of the matrix, in later rounds up to 60 times the wash volume was used. The amount of F-RNA remaining on the matrix after washing was detected and quantified by scintillation counting. The binding value was expressed as a percentage of the F-RNA used.

elution

In rounds 1-9, the binding F-RNA was non-biotinylated in three steps Peptide eluted. This was usually a 10-fold excess of the non-biotinylated used against the biotinylated CGRP and first one, then three hours and finally eluted overnight at 37 ° C. The eluted F-RNA was treated with phenol-chloroform Isoamyl alcohol extracted, precipitated with ethanol and resuspended in water. The one after these RNA affinity elution steps remaining on the matrix were not eluted but amplified directly on the matrix (see below).

From round 10, only one step eluted under denaturing conditions. For this purpose, the F-RNA remaining on the matrix after washing was added in two steps Incubate 100 µl of 8 M urea for 5 minutes at 65 ° C. The eluted F-RNA was extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol and in water added.

C. Amplification - Enzymatic Reactions Transcription - Creation of fluorinated RNA for use in selection

Transcriptions were made with 150 U T7 RNA / NA polymerase and the buffer supplied by the manufacturer (40 mM Tris-HCl pH 7.5; 10 mM NaCl; 6 mM MgCl ₂ ; 2 mM spermidine; 10 mM DTT) in a total volume of 100 µl carried out. MgCl ₂ was generally added to the reactions to a final concentration of 11 mM. The final concentrations of ATP and GTP were 1 mM each throughout the selection; 2'-Fluoro-UTP and 2'-Fluoro-CTP were used up to the 9th round with a concentration of 3 mM each, from the 9th round only with 1.5 mM. Between 20 and 50 µmol templates were used per 100 µl reaction, ie double-stranded DNA templates in the first round and in all subsequent rounds for double-stranded DNA, which was generated by enzymatic processing of the F-RNA selected in the previous cycle. ³² [P] -α-GTP was used for the radioactive labeling of the fluorinated RNA. The reactions were incubated overnight at 37 ° C and DNase I was added to digest the template. The F-RNA generated was then separated from non-incorporated NTPs under denaturing conditions using an 8% polyacrylamide gel with 8 M urea. The transcribed F-RNA was eluted from the gel, precipitated with ethanol, dried and taken up in pure water.

Reverse transcription - Generation of cDNA from selected F-RNAs

Reverse transcription of eluted fluoro-RNA was carried out using Superscript II and Buffer conditions of the manufacturer in a volume of 20 µl with 200 units enzyme and a dNTP concentration of 1 mM each. Usually, up to 8 µmol of the eluted F-RNA dissolved in 10 µl water and with 2 µl of a 100 µM solution of reverse primer mixed. This template-primer mixture was kept at 94 ° C. for 2 minutes denatured, transferred to ice and then in a thermal cycler to a temperature of 50 ° C set. After 2 minutes at 50 ° C 8 ul of a mixture of buffer, dNTPs and Enzyme added and the sample for 15 minutes at 50 ° C, another 15 minutes at 55 ° C and finally incubated at 68 ° C for 10 minutes.

In rounds 1-9, the F-RNA remaining on the matrix after the affinity elution reverse transcribed directly on the support and amplified with the help of PCR. Therefor the Qiagen RT-PCR kit was used.

PCR - Creation of the double-stranded DNA template

The cDNA generated in the reverse transcription was used directly in the PCR. there 6.66 µl of the 20 µl RT mix served as a template for a 100 µl reaction with 5 units Taq DNA polymerase. The concentration of the primers was 2.5 µm. The DNA was first denatured for 2 minutes at 94 ° C and then with a PCR profile from Amplified for 30 seconds at 94 ° C, 30 seconds at 55 ° C and 30 seconds at 72 ° C. The The number of cycles was generally kept as low as possible and usually amounted to on about 5-10 cycles.

results

The course of the selection is shown in Fig. 2. The values shown in FIG. 2A represent the CGRP concentrations used in the respective round and the associated percentage connection of the F-RNA pool.

Only the strand that ultimately produced the following sequences is shown. In most of the rounds, the binding was carried out with different peptide concentrations and a control selection without peptide (not shown). In general, the selected F-RNA of the most stringent strand, ie the lowest peptide concentration, which still gave a significant signal above the zero control, was processed as a template for the next round. Figure 2B is a graphical representation of this data.

The first round of selection was carried out with 2.5 nmol F-RNA molecules, the complexity was on the order of approximately 2.82 × 10 ¹⁴ different sequences. Since each sequence was only represented about five times under these conditions, a relatively high percentage of selected sequences was transferred to the second selection round in the first round. Between the 2nd and 5th round, between 0.36% and 2.65% of the F-RNA used was used in the subsequent round. During these five rounds, stringency was tightened by reducing the biotinylated D-CGRP offered for binding from 33 µM to 3.33 µM. In the 6th round there was a slight increase in the connection, which continued with a value of 12.8% of the F-RNA used in the 7th round.

By continuously reducing the CGRP concentration in the subsequent rounds, the stringency was increasingly tightened, which led to a breakdown in the binding of the F-RNA used to the peptide. A continuous increase in binding values in rounds 8 and 9 to 5.14% in round 10 correlated with an increase in the pre-selection (not shown) and was therefore not attributable to peptide-mediated binding. For this reason, the matrix was changed from neutravidin-agarose to streptavidinparamagnetic particles in round 11. This was reflected in a drop in the percentage connection to 1.23%. However, a peptide-mediated increase to 5.74% was recorded in round 13. Over the next three rounds, a value of about 3.5% binding was established at a CGRP concentration of 125 nM. Since this value did not increase in rounds 15, 16 and 17 (round 17 not shown) despite the same stringency, rounds 17 and 18 were carried out as a so-called double round. The reasoning for this methodical approach is based on the assumption that a stagnation of the percentage binding with a constant peptide concentration is the result of a kind of balance between the actual binding and the subsequent amplification. In order to shift this balance in favor of binding, the F-RNA was subjected to a double binding process: the F-RNA was first bound at the less stringent CGRP concentration of 1.25 μM, then eluted and purified. The F-RNA collected in this way was now not processed by enzymatic amplification as usual, but refolded and used directly in a further round of selection under more stringent conditions. As can be seen from FIGS. 2A and B, this method was able to achieve 4.89% and 31.51% binding of the F-RNA used (as can be seen from FIG. 2A) in round 18 at peptide concentrations of 1 and 10 nM , This F-RNA was reverse transcribed and amplified by PCR. The DNA was cloned and a total of 192 clones were sequenced.

sequences

The result of the sequence analysis is shown in FIG. 3. The sequence shown in italics (third sequence from above) is that of clone 732. Mutations are shown in bold, the contribution of the primer binding site is marked by underlining. The following order was found for the different clones:

Of the 192 clones, both primers could be found in 180 clones. Of these 180 Sequences, a sequence with 168 copies was clearly the most frequently represented. The The remaining twelve sequences differed from the main sequence only by Point mutations and base deletions: One point mutation came five times, five more once before. A deletion of two nucleotides occurred twice.

A comparison of the binding strength of these sequences was made using the Biacore device and found that seven of the eight sequences show the same affinity for the target molecule. The K _{D of} the clone shortened by two nucleotides was measured at approximately 10 nM. The binding constant was two times higher at 37 ° C. Binding constants were determined using both the Biacore device and isothermal calorimetry (ITC). The calorimetry experiments were carried out at 37 ° C. in degassed selection buffer with a stirring speed of 300 rpm. The measuring cell was filled with a 10 µM solution of the respective 2'F Spiegelmer. The injection syringe contained a 25 µM solution CGRP, which was injected into the measuring cell after an initial 5 µl injection in 7.5 µl fractions. The injection process lasted 10 s in each case, the time interval between two injections was 300 s.

The Biacore experiments were carried out at 37 ° C in degassed selection buffer. On the streptavidin chip was immobilized with biotinylated CGRP (blank - 140 RU - 640 RU - 900 RU). Spiegelmer's solution in a concentration of 500 gave a typical one Binding signal, which is evaluated with standard Biacore software (1: 1 binding model) has been.

Example 2 Shortening of 2'-F-RNA aptamers that bind free D-CGRP

Starting from the D'-CGRP binding 2'-F-RNA aptamers described in Example 1 and the corresponding 2'-F-RNA level meres that recognize free L-CGRP tried to shorten the length of the respective aptamers or Spiegelmers. Of the total of eight clones, as shown in Example 1, seven clones were distinguished by similar binding constants from 10 to 50 nM. Clone 670 recognized D-CGRP significantly worse. As expected, the selected aptamers, the D- Oligonucleotides obtained from cloning and sequencing accordingly Plasmids were expressed via an intermediate PCR step, natural L- CGRP does not, as measurements on the biacore have shown.

The secondary structure of some of the aptamers and Spiegelmers described in Example 1 with the rnafold program (LL. Hofacker, et al., 1994, monthly Chem. 125, 167-188) certainly. In particular, the secondary structure models of the Aptamers 666 and 732 as well as the Spiegelmere 732-029, 732-026, 732-100 and 732-108. The results are in Figs. 4 and 5 and for the Spiegelmers in Figs. 6, 7, 8 and 9. The Secondary structure models agreed well with the results of enzymatic probing Experiments (Ehresmann, C., et al., 1987, Nucleic Acids Res 15 (22): p. 9109-28).

The secondary structures each consist of a trunk, two hairpin bows and a 15- or 16-nt bulge and thus form a trefoil-like structure. The bulges in the structural core, formed by C (10) -C (11) -U (12) and C (54) -U (55) -C (56), and the hairpin loop U (13) to A (26) seem essential for the detection of the Target molecule to be CGRP. Aptamers binding for CGRP or L-CGRP binding Spiegelmere can thus capture a minimal binding motif, which is a Cloverleaf-like structure. An alternative minimal binding motif is that Formation of a strain with two hairpin bows and a 15 or 16 nucleotides extensive bulge. The bulge is preferably formed in the structural core by the nucleotide sequence C (10) -C (11) -U (12) and C (54) -U (55) -C (56) and the Hairpin loop through the nucleotide sequence U (13) to A (26), as shown in Figs. 4 to 9 shown.

Due to the measured binding affinity to the target molecule CGRP with a K _d of 10 nM, clone 666 was selected as the starting clone for the shortening and optimization of 2'-F-RNA aptamers and Spiegelmers. As described in Example 1, clone 666 was isolated from an N40 pool, ie a pool whose randomized sequence has a length of 40 nucleotides, and consisted of a 79-nt sequence. The sequence has a particularly striking region between nucleotide positions 41-50, in which 7 out of 10 nucleobases are uraciles. The secondary structure of clone 666 was determined with the program rnafold IL Hofacker, et al., 1994, monthly. Chem. 125, 167-188 predicted and is shown in Fig. 4. In this case, too, the secondary structure prediction was in good agreement with the results of enzymatic probing experiments (Ehresmann, C. et al., 1987, Nucleic Acids Res. 15 (22), 9109-28). The aptamer forms a characteristic cloverleaf-like structure, consisting of a stem with several small bulges, two hairpin bows (I and III) and an apparently unstructured 16-nt bulge (II) that connects the two hairpin bows. All four structural elements have their origin in two opposite bulges, each consisting of three bases. The primer binding sites, especially the 5 'primer binding site, are integrated in the strain.

For the synthesis of 2'-F-RNA Spiegelmeres it was necessary to sequence clone 666 shorten and optimize to the extent that the resulting minimal binding motif is a chemical synthesis with reasonable effort allows while maintaining the high Affinity for the target molecule.

A minimal binding motif was created using direct Nucleotide point mutations and deletions identified. A series of deletions with Tetranucleotide blocks gave an initial overview of regions that are responsible for the binding of the Target molecule are essential. Deletions in all three double helix regions were possible and did not significantly reduce binding affinity, but only as long as a tribe formation was still possible. As soon as too many deletions are lost the formation of the strain no longer bound to the target molecule CGRP observe. This was observed when the closing stem of the nucleic acid binder either on less than five base pairs or the hairpin loops I and III on less than 3 base pairs were shortened.

Deletions in the three loops led to different results.

Each of the embodiments of the following sequences described below represents a nucleic acid according to the invention. Cutting out the nucleotides G (20) - A (21) in hairpin loop I (in analogy to clone 732) led to a nucleic acid binder which was still highly affine (K _d = 20 nM), however, any further deletion or mutation tested in loop 1 led to a complete loss of binding. In loop III, nucleotides A (46) and U (47) could be removed without loss of binding. As expected, the deletion of one of the bases U (48), U (49) or U (50) in combination with A (46) led to the same result. Various shortenings were also possible in Loop II, in particular the removal of the bases G (28), U (29), A (30), U (31), A (33) and A (35) was tolerated, but it did to different degrees of change in binding affinity. A combination of these deletions was only possible in the case of the co-deletion of U (29) -A (30). The stem of the hairpin loop I allowed a further shortening of the stem length to three base pairs when deleting the base pair A (15) and U (24) (the stem length is already in the loop when deleting the nucleotides G (20) -A (21) I shortened from 5 base pairs to 4 base pairs (clone 732, Fig. 5)). The bulges C (10) to U (12) and C (54) to C (56) seem to be essential for the recognition of the target molecule or for the tertiary structure required for this, since the deletion of one of these nucleotides already leads to the complete loss of the recognition of the target molecule CGRP leads.

In summary, it can thus be stated that in particular the loop of the Hairpin bow I and the central bulge of the cloverleaf structure essential for the Binding of the target molecule. The hairpin bow III, the smaller bulges in the The center of the cloverleaf structure and the adjoining stem probably serve the Stabilization of a special tertiary structure.

Further reductions were possible with the almost complete removal of the 3 'primer binding site C (64) to C (79) and the removal of the dinucleotide A (3) -G (4), a small part of the 5' primer binding site from the final strain of Nucleic acid binders can be achieved. Such a shortening is represented by the clone 732_029. This shortening was accompanied by only a slight loss of affinity (K _d = 30 nM). The final strain could be further reduced to five base pairs by the deletions of the base pairs C (5): G (61) and U (6): G (60) (K _d = 30 nM, as is also described in FIG. 7) ).

Testing different combinations of these different deletions resulted in clone 732_100 (length 51 nt, K _d = 75 nM, Fig. 8). The deletions A (3) -G (4), C (5): G (61), U (6): G (60), G (20) -A (21), A (15): U (24), A (46) -U (47) and C (64) to C (79) combined. This 51mer binds to the target molecule CGRP with a K _d of 75 nM. However, an analysis of the structure predicted by the rnafold program shows a potential alternative folding, which is caused by a mutation of the base pair C (14): G (25) to G (14): C (25) (clone 732_103) or the base pair C ( 44): G (52) after G (44): C (52) (clone 732_104) can be prevented. In fact, these two clones also show an increased affinity for CGRP with a K _d of 20 and 40 nM, respectively. Clone 732_104 can be further combined with the deletion G (32), whereby clone 732_108 is obtained, a 50mer with a K _d of 35 nM ( FIG. 9).

The sequences 732_026 and 732_029 were selected for the production of Spiegelmer to determine their biological activities. Since no solid phase material with L-2'-F-modified C was available, it was first tested whether mutation of each GC base pair terminal in a CG base pair corresponding to the clones 732_045 or 732_096 (Fig. 10 and Fig. 11 ) was possible without a measurable loss of binding affinity. Since almost identical K _d values were obtained for 732_045 and 732_096 compared to 732_026 and 732_029, they were selected for the production of Spiegelmer to determine their biological activities.

Another method to find a minimal binding motif was followed when clone 666 or truncated forms thereof were combined from two independently synthesized fragments. In two preliminary tests, fragmentation was tested in one of the two hairpin loops I and III. On the one hand G (1) to C (19) and A (22) to C (79) (shown with clone 732_070a and 732_070b, Fig. 26) and on the other hand G (1) to A (46) + AG and C + U (49) to C (79) (shown with clone 732_071a and 732_071b, Fig. 27).

The composite system 732_070ab showed no measurable binding affinity for CGRP. This result reinforces the assumption that hairpin loop I is involved in the binding process with CGRP, as is also suggested by data from the enzymatic probing experiment. The fragments 732_071a + 732_071b, on the other hand, form a nucleic acid binder _that binds to the target molecule CGRP with a K _d of 100 nM. Further experiments showed that the strain of the former hairpin loop III had to be at least five base pairs long in order to obtain a measurable binding event. Assembling a nucleic acid binder from two fragments is an efficient alternative to obtain a minimal binding motif. It allows the synthesis and production of relatively long nucleic acid binders on a large scale with a reasonable scientific and also economic effort. The same principles apply to the generation of composite CGRP-binding nucleic acid structures as for one-piece nucleic acid binders. The affinity and bioactivity of the resulting nucleic acid binder 732_071ab according to the invention was comparable to that of the one-part nucleic acid binders 732_108, 732_096 and 732_045 in the measured areas in the assays used (732_071ab, K _d = 100 nM), 732_108 K _d = 35 nM _d = 732_096 30 nM and 732_045 K _d = 30 nM).

The Spiegelmer / L-CGRP and aptamer / D-CGRP complexes resulted within the experimental error deviation matching equilibrium binding constants.

Nucleic acid binders 732_045, 732_096 and 732_071ab were selected for further biological studies and in the corresponding enantiomeric forms synthesized.

The various sequences created and used in this example are shown in FIG . The sequences shown in FIG. 18 can be followed by the following SEQ. ID. No. be assigned:
sequence SEQ. ID. No. 666 9 732 10 732_026 11 732_029 12 732_045 13 732_096 14 732_100 15 732_103 16 732_104 17 732_108 18 732_070a 19 732_070b 20 732_071a 21 732_071b 22

Example 3 Selection of 2'-F-RNA aptamers

A further selection was carried out with the aim of identifying 2'-F-RNA aptamers against D-CGRP. The reaction conditions used are the following:
The target molecule used is CGRP from rats. Sequence and provision took place analogously to the procedure described in Example 1.

Selection pool, creation of the start pool

The selection pool SK60 consists of a randomized region of 60 nucleotides, which is flanked by the T7 primer (37 nucleotides) from the 5 'end and by the reverse primer (20 nucleotides) at the 3' end. The T7 primer contains a transcription initiator and a forward primer area. The forward primer begins with a guanosine triplet to improve transcription efficiency.
SK60 Pool: 5'-GGG AAT TCG AGC TCG GTA CC-N ₆₀ -CTG CAG GCA TGC AAG CTT GG-3 '
SK.60T7: 5'-TAA TAC GAC TCA CTA TAG GGA ATT CGA GCT CGG TAC C-3 '
SK.60F: 5'-GGG AAT TCG AGC TCG GTA CC-3 '
SK.60R: 5'-CCA AGC TTG CAT GCC TGC AG-3 '

The annealing temperature for the primers was calculated theoretically and then experimentally optimized within the calculated framework.

T _m = melting temperature, T _p = 22 + 1.46 × [2 × (#GC) + (#AT)] = optimal annealing temperature with optimal amplification, according to Wu et al., 1991, DNA and Cell Biology 10, 233; without T7 promoter region.

The pool was produced synthetically, the base composition determined and a complexity of 1 × 10 ¹⁵ molecules corresponding to 1.78 nmol ssDNA amplified via polymerase chain reaction (PCR). 1.78 nmol of the double-stranded DNA was transcribed into 2'-fluorinated RNA according to protocol 1 in the in vitro transcription with fluorinated pyrimidine nucleotides (TriLink BioTechnologies, San Diego, CA92121). The 2'-F-SK60 RNA start pool thus produced was used in the first selection round. Protocol 1 Approach of in vitro transcription for the 1st round

selection buffer

The selection buffer (20 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ and 1 mM CaCl ₂ ) is based on the physiological conditions of human blood and is used throughout the selection. The pH of 7.4 was set at 37 ° C.

Selection strands and stringencies

Two different selection approaches were carried out to identify a CGRP-binding 2'-fluoro-RNA, the characteristics of which are summarized in the following table. Tab. Characteristics of the selection approaches against rCGRP-1

The first approach was made using neutravidin agarose (Pierce) as a matrix Immobilization of the 2'-F-RNA / peptide complex carried out. The bound 2'-fluoro- RNA was eluted from the matrix in 2 steps. First, the trusses were over Affinity elution by competition with an excess of non-biotinylated CGRP eluted. This was followed by elution with 8 M urea by denaturing the 2'- Fluoro-RNA-peptide complex. The eluates were amplified separately worked up to limit a selection pressure on the part of the enzymatic reactions.

The second selection approach was based on steptavidin-derivatized magnetic particles Matrix for immobilization of the 2'-fluoro-RNA peptide complex carried out. The elution was also carried out with 8 M urea by denaturing the RNA / peptide complex.

Folding the 2'Fluoro-RNA

In order to obtain a Mg ²⁺ and Ca ²⁺ ion-independent folding of the 2'-fluoro RNA, the denaturation and renaturation (folding) was carried out in selection buffer without Ca ²⁺ and Mg ²⁺ ions. The 2'-fluoro-RNA was denatured in selection buffer without Mg ²⁺ / Ca ²⁺ for 5 minutes at 95 ° C. and then folded at 37 ° C. for 30 minutes. The ions were then added and the mixture was folded for a further 10 minutes at 37 ° C. The batch was placed directly on a guard column.

precolumns

The guard column was used for counter-selection of potentially matrix-binding 2'F-RNA molecules performed and thus generally serves to deplete matrix binding 2'-F-RNA within the selection process. A guard column is to be understood an unloaded column, which is the actual peptide column within a selection round is connected upstream. The guard column was dimensioned the same as the main column. The 2'-F-RNA was incubated for 30 minutes at 37 ° C with the matrix, and then the 2'-F- RNA was washed from the matrix with a column volume of selection buffer. The on the Labeled RNA remaining in the matrix was determined and documented as a precolumn. The eluted 2'-F-RNA was further used for the selection process.

The course of the guard columns during the selection, ie the portion of labeled RNA remaining on the matrix, is shown in FIG. 12. It is noteworthy that from round 13 the selection SA was divided into two selection strands, namely selection strand SA-1 and SA-0.1, which differed in the peptide concentration. In the case of SA-1, the peptide concentration in round 13 was 10 nM and in rounds 14 to 16 1 nM, whereas in the selection approach SA-0.1 the concentration of peptide in round 13 was 10 nM and then in rounds 14 to 16 was only 0.1 nM.

Connection and immobilization

The biotinylated D-CGRP was added directly to the 2'F-RNA in solution. The concentration of the biotinylated peptide in the binding mixture was successively reduced in the course of the selection process, as shown in the table below. Tab. Reduction of the peptide concentration

The connection of the biotinylated rCGRP-2'-F-RNA approach to the neutravidin or Steptavidin-derivatized matrix was done by directly adding the binding approach to the Matrix. Binding was carried out in a thermal shaker (Eppendorf) for 10 minutes at 37 ° C a shaking speed of 800 rpm.

To wash

The bio-CGRP-2'F-RNA complex immobilized on the matrix was washed with selection buffer in order to remove unbound 2'F-RNA from the column in individual washing steps. CGRP-1 binding 2'F-RNA remains on the matrix. A wash volume was 100 µl selection buffer. During the selection, the stringency of the selection was increased by increasing the washing steps, as can also be seen from the table below. Tab. Course of the washing steps

Elution of the bound 2'-fluoro-RNA Selection approach NA-A

Elution of the 2'-F- bound to the matrix via the peptide RNA was first carried out by competition with non-biotinylated CGRP in a 10-fold Excess over the immobilized bio-CGRP. The incubation was carried out for 12 hours 37 ° C in the overhead shaker. The remaining 2-F-RNA was then eluted by denaturing the 2'-F-RNA / peptide complex by adding 8 M urea. The Incubation took place at 65 ° C for 20 minutes in a thermal shaker 1200 rpm shaking speed.

Selection approach SA-1 / 0.1

The elution was carried out by denaturing the 2'-F-RNA / peptide complex by adding 8 M urea. Incubation was at 65 ° C for 20 minutes in a thermal shaker at a shaking speed of 1200 rpm.

The course of the eluted 2'-F-RNA and the peptide concentration of the selection approach NA-A is shown in FIG. 13. An accumulation of binding 2-F-RNA was shown in the sixth round of selection at 1 µM peptide concentration. The peptide concentration was continuously reduced to 10 nM. The peptide concentration was reduced only after renewed enrichment of binding 2'-F-RNA at a given peptide concentration, as shown in rounds 6, 8, 10 and 13. The selection was plateaued from rounds 13 to 16 at 10 nM peptide, and the enriched nucleic acids were cloned and sequenced.

The course of the eluted 2'F-RNA and the peptide concentration of the selection strands SA-1 and SA-0.1 is shown in Fig. 14 A / B. The selections SA-1 and SA-0.1 were carried out together until the 13th round as a selection strand and then into the different selection strands SA-1 and SA-0.1 with different continuation of the peptide concentration up to round 16, cloned and sequenced. The peptide concentration was reduced only after renewed enrichment of binding 2'-F-RNA at a given peptide concentration, as shown in rounds 8, 11 and 13.

amplification Extraction and precipitation

The eluted 2'-F-RNA was purified by means of phenol-chloroform extraction and then with glycogen as carrier, 0.3 M sodium acetate pH 5.5 and 3 volumes 50:50 (V / V) ethanol-isopropanol precipitated at -80 ° C for 30 minutes.

Reverse transcription (RT)

The precipitated 2'-F-RNA was converted into single-stranded DNA (ssDNA) by means of reverse transcription rewritten. <5 pmol of 2'-F-RNA template are used per 40 µl batch.

First, the 10 µM SK60-reverse primer was annealed after denaturing (5 minutes 95 ° C) the 2'-F-RNA template in the presence of 0.8 M betaine by directly cooling the sample on ice (so-called snap cooling). The first strand buffer (250 mM Tris / HCl, pH 8.3, 375 mM KCl, 15 mM MgCl ₂ ), 10 mM DTT and deoxyribonucleic acid is then added. The buffer conditions correspond to those for the enzyme Superscript II (standard conditions of the manufacturer). The mixture is heated to 48 ° C. for 2 minutes and 5 units of the reverse transcriptase are added. The incubation was carried out via a temperature step gradient (48 ° C. 30 minutes, 50 ° C. 20 minutes, 55 ° C. 10 minutes, 70 ° C. 15 minutes) in a PCR thermal cycler.

Polymerase chain reaction

10 µl each of the RT mix were used as templates for the PCR, which corresponds to a template amount of 1-5 µmol / 100 µl PCR mix. The nucleotide sequences of the primers are:
SK.60T7: 5-TAA TAC GAC TCA CTA TAG GGA ATT CGA GCT CGG TAC C-3 '
SK.60R: 5'-CCA AGC TTG CAT GCC TGC AG-3 '

The PCR mixture was combined as follows and run to a yield of dsDNA of approx. 1 pmol / μl mixture with 1 minute of 94 ° C. denaturation, 1 minute of 72 ° C. annealing and 1 minute of 72 ° C. elongation. The reaction was analyzed on a denaturing 8% polyacrylamide gel. Protocol 2 PCR reaction during selection

The PCR batches were precipitated with ethanol, resuspended in water, and 50-100 pmol Material was used for in vitro transcription.

2'-fluoro transcription

50 pmol dsDNA were used as a template for the transcription with fluoro- Pyrimidine nucleoside triphosphates used. The ratio of the 2'-floor Pyrimidine nucleoside triphosphates to the unmodified purine nucleoside triphosphates 3: 1. The incubation was carried out overnight at 37 ° C. in a thermoblock.

4. Results Course of the selections

The selections were created in the first round as a collective round, if possible none losing binding F-RNA molecules. In the following rounds, the Intensification of the washing steps, the reduction of the peptide concentration, as well as the Increasing the RNA: peptide ratio the stringency with regard to the selection of high affinity binding 2'-F-RNA continuously grown.

A first enrichment of CGRP-binding 2-F- RNA will be shown in rounds 6 and 8. After increasing the stringency factors and subsequent drop in the signal, a renewed increase in binding 2'-F- RNA can be observed. The selection was finally made at a peptide concentration of 10 nM plateaued for the signal, and the eluates (affinity elution and denaturing elution) were separately amplified, cloned and sequenced. NA-A denotes the sequences of the eluates of the affinity elution and NA-D the eluates of the subsequent denaturing elution.

The selection strand SA showed an accumulation of CGRP-binding 2'-F-RNA in round 8. A reduction in the peptide concentration always led to a drop in the Signals with subsequent enrichment (rounds 11 and 13). After round 13 the selection strand was split into the two strands SA-1 and SA-0.1. SA-1 was at a peptide concentration of 1 nM, SA-0.1 at a peptide concentration of 100 pM in a Signal plateau brought; both strands were then separately amplified, cloned and sequenced.

sequences

The following sequences were obtained as part of the selection: families

Tab x.

The sequences mentioned above correspond to the following SEQ. ID. No .:
sequence SEQ. ID. No. 1 23 2 24 3 25 4 26 5 27 6 28 7 29 8th 30 9 31 10 32

ranking

A ranking of the clones was determined using binding experiments against 1 µM D-CGRP in solution. The folded, radioactively labeled RNA was incubated with the peptide at 37 ° C. for 2 hours, immobilized on neutravidin agarose, washed, and the percentage binding of the 2'-F-RNA to CGRP was determined. Clones D7, El, G10 and E3 showed the highest percentage connection. The result is also shown in Fig. 19. The assignment of the origin of the clones from the respective selection strands NA-A (affinity elution), NA-D (denaturing elution), SA-1 and SA-0.1 is shown under the x-axis.

competition

Competition tests were carried out to further characterize the clones C5, E1, F1, D7, B3 and F10. The result is shown in FIG. 20. Competition from non-biotinylated D-CGRP was determined by incubating the biotinylated D-CGRP (1 µM) and the free CGRP (10 µM) at 37 ° C with radioactively labeled 2-F-RNA for 2 hours. It could be shown that clones C5, E1 and D7 can be competed for by free CGRP, whereas clones F1, B3 and F10 do not recognize the non-biotinylated CGRP.

Bead assay to determine the dissociation constant

50 µmol each of the labeled 2'-F RNA from clones E1, D7 and 732 (reference, origin the 2'-F-RNA selection with pool PB40) were folded and used for 3 hours different bio-CGRP concentrations in solution at 37 ° C in a thermoshaker incubated. Then a constant amount of magnetic steptavidin beads (Roche) added as a matrix and the 2'-F-RNA / peptide complex for 10 minutes at 37 ° C and a shaking speed of 800 rpm. The magnetic Particle matrix was separated using a magnetic separator, the supernatant removed and the difference of bound / unbound 2'-F-RNA was determined. Of the identified The control (0 µM bio-CGRP) was subtracted as background. The determined Values were entered into the GraphFit software program (Erithacus Software Ltd.) and the dissociation constant is determined.

The estimated dissociation constant is then 196 nM for E1 and 42 nM for D7. The Reference clone 732 (see application example 1) from the selection with the start pool PB40 as an internal control for the bead assay used here resulted in a dissociation constant of 23 nM. The result is shown in Figs. 21 and 22. Other sequences obtained are shown in Figs. 48 and 49 shown.

Example 4 Use of nucleic acids without primer binding sites for the selection of D-CGRP binding nucleic acids

The following materials were used for the investigations described herein, the details of the manufacturers of the following substances, solutions and Enzymes were listed in the corresponding text passages. In all cases LiChromosolv water used by Merck.

Rats α-D-CGRP was synthesized by Bachem, Heidelberg. That for selection The peptide used carries a biotin group at the carboxyl terminus around the separation of unbound nucleic acids using the biotin streptavidin or biotin neutravidin Enable connection. Neutravidin-Agarose and Ultralink Plus were used as the matrix Immobilized streptavidin gel (both from Pierce) used.

The oligonucleotides used, i.e. H. RNA and DNA nucleotides, such as primers, start pools, Ligation constructs and the like were all part of NOXXON Pharma AG Standard phosphoramidite chemistry synthesized. The sequences can be found in the following Overview.

Oligonucleotides used and tested STAR-1 oligonucleotides

For the sequences disclosed herein, the following applies with regard to the spelling:
Information in italics denotes nucleotides of the ligation matrices that hybridize with the library.

The underlined area of a sequence means that this area corresponds to the T7 RNA Corresponds to polymerase promoter.

N _OH generally denotes a ribo-nucleotide.

C _Ome denotes a 2'-OMethyl modified cytosine.

P denotes a 5'-phosphate overview of the different sequences

Library

Forward

Reverse

STAR-1 Reverse Matrix corresponds to STAR-1 Reverse Primer Reverse Ligate

Production of the start pool

For a complexity of 1 × 10 ¹⁵ molecules, 1.67 nmol start pool were introduced into the selection. Correspondingly, 33.4 transcriptions with 50 µmol "STAR-1 initial pool, reverse strand" (5 'C _OMe C _OMe T GTC- (dN) ₄₀ -GTC CTA TAG TGA GTC GTA TTA GTA GTG CGA AG) were used as a template for a 100 µl reaction used. To achieve a DNA double strand in the region of the T7 promoter, "STAR-1 forward primer" was added to the reaction mixture in a 1.5-fold excess of "STAR-1 initial pool, reverse strand". The RNA was then gel purified and precipitated (see below for transcription and gel purification).

selection steps Denaturation and folding of the RNA

All non-enzymatic steps of the selection - with the exception of denaturation - were in selection buffer (10 mM Hepes-KOH pH 7.5 (Biochrom AG); 150 mM NaCl; 4 mM KCl; 1 mM MgCl ₂ ; 1 mM CaCl ₂ (all Ambion) and 0.05% Tween-20 (Sigma)). The denaturation was carried out for 5 minutes at 95 ° C. in selection buffer without Tween-20, MgCl ₂ and CaCl ₂ . After denaturation, the RNA was first cooled to 37 ° C (or room temperature) for 15 minutes at 37 ° C (or room temperature). Tween-20, MgCl ₂ and CaCl ₂ were added and folding continued again at 37 ° C (or room temperature) for 15 minutes.

connection

Following the folding, the RNA was initially at 37 ° C (or room temperature) for 30 minutes without peptide either with the Neutravidin-Agarose matrix or with Ultralink Plus Immobilized streptavidin gel incubated. This so-called preselection served the Separation of potential matrix binders. After this incubation step, the unbound RNA separated from the matrix by filtration through MobiTec columns, with different concentrations of biotinylated CGRP and for at least 2 Hours - at low peptide concentrations, if necessary also overnight - at 37 ° C (or room temperature). Thereupon the binding approach became the biotin binding Matrix added. The matrix with the RNA bound to it was removed after 30 min Solution separated by centrifugation and washed with selection buffer. That here wash volume used in the first rounds was 5-10 times the amount of the matrix, in later selection rounds with 25 times the wash volume.

Elution of the binding RNA molecules

For this, the RNA remaining on the matrix after washing was checked twice with each 400 µl of 8 M urea with 10 mM EDTA (both from Ambion) for 15 minutes at 65 ° C from Matrix material eluted. The eluted RNA was 400 ul Phenol / (chloroform / isoamyl alcohol) mixture (1: (1: 1/24)) (Applichen) added, 5 min Centrifuged at 13,000 rpm at room temperature, the aqueous phase (supernatant) removed, the phenolic phase was re-extracted once with 100 ul water, the aqueous phases combined and shaken with 500 µl chloroform-isoamyly alcohol mixture (24: 1) (Applichen), 5 min Centrifuged at 13000 rpm to room temperature and the upper aqueous phase removed.

The aqueous phase was then treated with 2.5 times the volume of 100% ethanol (Fluka), 0.3 M Sodium acetate (Ambion) and 1 µl glycogen (Roche) precipitated for 30 min at -80 ° C and for 30 min Centrifuged at 14,000 rpm (4 ° C). The pellet was once ice-cold 70% Washed ethanol (Fluka).

Ligation and amplification Preparation for the ligation

The decisive factor for successful ligation was the determination of the RNA to be ligated Quantity. The amount of RNA eluted was determined via the radioactivity and - if possible - via the OD determines. The disadvantage of OD determination is that a minimum volume of 50 µl is necessary. For the ligation approach, however, the RNA was not allowed to be in too large a volume be included.

Therefore, at the beginning of each selection round, the specific activity [cpm / pmol] certainly. This made it easy to estimate the amount of eluted RNA.

Estimate the amount of H ₂ O used to dissolve the pellet

Since the ligation approaches were optimized for template amounts of up to 5 pmol template or from 5 pmol template, the corresponding protocol was chosen for the following calculation of the H ₂ O amount. The maximum amount of H ₂ O was determined using the ligation protocol and the pellet was dissolved accordingly.

ligation

In preliminary experiments, two approaches with different (template amount) / (reaction volume + enzyme amount) ratios were determined for the ligation. The approaches also vary if, instead of the "simultaneous ligation" listed here, the "parallel 2-step ligation" was carried out (see below). Batches up to 5 pmol: "Simultaneous ligation" Volume of the batch: 14 µl per 1 pmol RNA template

"Parallel-2-step ligation"

In this case the dissolved RNA was divided into two batches. The first approach was first ligated at the 5 'end, while the second approach was first ligated at the 3' end. Accordingly, only the appropriate amount "ds F or R adapter" was initially added to the batch. After the first ligation step, the missing "ds F or R adapter" was then added accordingly, 5 min. heated to 50 ° C and then again RNAse-out and ligase added. Batches from 5 pmol: "Simultaneous Ligation" Volume of the batch: 14 µl per 5 pmol RNA template

"Parallel-2-step ligation"

(as described above)

Ligation after the first round

In contrast to the protocol described above, after the 1st round the addition of ds R adapter 2 (for n + 1 transcripts) is not necessary because the transcription is before the first round was terminated properly by using two terminal 2'-O-methyl nucleotides. Therefore, after the first round, only the R adapter 1 (Rev Ligat / Rev Primer) used in 20 × excess to the eluted RNA.

Reverse transcription

The reverse transcription took place - directly after the ligation - with up to 15 µmol ligated RNA in a volume of up to 100 ul. For larger quantities of material several parallel approaches are made. The size of the rT approach is due to the differently concentrated ligation approaches determined (simultaneous ligation / parallel-2 Ligation step up to or from 5 µmol).

Key points for the RT were:

The temperature conditions were: 20 min at 51 ° C + 10 min at 54 ° C; then 4 ° C

PCR

PCR was carried out with a maximum of 1 µmol template per 100 µl PCR. If a bigger one RNA was eluted, several PCRs had to be set up in parallel. If considering that before the start of an increase only 1 µmol is often eluted and thus the Compexity cannot be greater than the number of molecules in a pmol without a doubt PCR-amplify even a part (> 1 µmol) of the cDNA (you don't have to insert the entire cDNA in PCR). This was done from round 5 onwards.

The reason for using a maximum of 1 µmol ligation template in the PCR is the Ligation matrix for the n + 1 transcripts (3'-micro heterogeneities in the transcription). The used ligation matrix with the "universal" base (2'-deoxyinosine) is in the PCR delayed and can inevitably prime there as a reverse primer. This is not how one arises fissile PCR product. The more template is brought into the PCR, the more Ligation adapters are carried over and the more unsplittable PCR product is created. Therefore, no more than 1 µmol template should be used in the PCR.

In contrast, if inosine is used as the "universal base" for the ligation matrix, up to 5 pmol (or more, not tested) template to be used in the PCR. In this case the PCR products primed through the ligation matrix are also alkaline digestible (was in this selection is not used). As with ligation and reverse transcription, there were two different PCR protocols for simultaneous ligation / parallel 2-step ligation up or from 5 pmol.

Key points for the PCR were:

As a rule, 13-15 PCR cycles were carried out.

Alkaline cleavage of the reverse strand and test gel

First, 10 µl of PCR product were cleaved by alkaline tests. The standard recipe for the analytical alkaline cleavage of the reverse strand was as follows:

The pH was then checked using indicator paper. The pH should be in be around pH 5-6.

The cleaved PCR product was tested using an ssDNA standard (length 78 nt) 3.55 µmol NaCl (Ambion) was added to compensate for the salt effect) on a 10% denaturing PAGE quantified to determine which volume is alkaline cleaved PCR product into the following in vitro transcription (target amount: 50 µmol PCR Product per 100 µl batch) must be used. As a rule, 100 µmol PCR Product digested alkaline (for two 100 µl in vitro transcriptions).

Ethanol precipitation with sodium acetate

The ethanol precipitation with sodium acetate (Ambion) was mainly used for Desalt the samples.

1/10 volume of the PCR was performed on 3 M NaOAc (pH 5.3), 2.5 times the PCR volume on 100% Ethanol and 1-2 µl glycogen (20 µg / ml) added. The mixture was at -80 ° C for 30 min precipitated, centrifuged for 30 min at 13,000-14,000 rpm at 4 ° C, the pellet once with 500 µl Washed 70% ethanol and centrifuged for 5 min at 13,000-14,000 rpm. The pellet was resuspended in water (1/10 of the original PCR volume) or the T7 The transcription batch was pipetted directly onto the pellet (and then resuspended).

T7 transcription

Radioactive alpha- ³² P-GTP (Hartmann) was incorporated during T7 transcription. 50 µmol template (per 100 µl mixture) was introduced into the in vitro transcription. As a special feature compared to other transcriptions, an 8-fold excess of guanosine 5'-monohosphate (8 × GMP) was added via GTP, so that most transcripts should have carried a 5'-monophosphate.

Key points for in vitro transcription were:

The 10 × Txn buffer (SK) contains 800 mM Hepes / KOH (biochrom), 220 mM MgCl ₂ (Ambion), 10 mM spermidine (Fluka), pH 7.5.

The in vitro transcription was carried out for 8-16 hours at 37 ° C. In connection the remaining DNA was digested with about 10 units of DNAse I (Sigma) (20 min at 37 ° C). The Digestion was stopped by adding EDTA (Ambion) (final concentration 25 mM).

Purification of the transcribed RNA

The 100 µl transcription batches were each with 50 µl concentrated urea solution (Roth) Blue marker (7.2 M urea, 10% bromophemol blue (Merck)) added, 5 min at 95 ° C denatured and placed on a preparative denaturing 10% PAA gel (running time about 1 Hour at 50 watts). An RNA (50mer) and DNA (78mer) size standard (each 250 µmol) applied. The bands were "UV Shadowing "(at 256 nm) made visible.

The cut out gel band was eluted using "Crush &Soak". For this purpose, the gel pieces were taken up in 360 μl H ₂ O and 140 μl 5 M ammonium acetate (final concentration approx. 2 M). The crush-and-soak elution was carried out 2 × 1.5 hours at 68 ° C. on the thermal shaker (1000 rpm). The supernatants were filtered through Ultrafree MC columns (Millipore) in the table centrifuge in 2 ml Eppis. The crush-and-soak filtrate (500 µl) was added 1-2 µl glycogen (20 µg / ml) and 1250 µl 100% ethanol, the RNA precipitated 30 min at room temperature, centrifuged for 30 min at 13,000-14,000 rpm, the Wash the pellet once with 400 µl ice-cold 70% ethanol and finally centrifuge for 5 min at 13,000-14,000 rpm. The dried pellet was taken up in 100 μl of water and the RNA concentration was determined photometrically at 260 nm.

Results Simultaneous 5 'and 3' ligation: STAR-R02

Selection STAR-R02 was divided into two strands in round 6. The first strand was led into the plateau with a peptide concentration of 10 μM and a 2-fold excess of peptide. Round 10 was cloned and sequenced. The result is shown graphically in FIG. 28 and in a table in FIG. 29.

Sequencing of selection round STAR-R02-10

62 different sequences were obtained from 82 evaluable sequences. From that 19 carry the characteristic core motif 1 (CATACGGTGAAAGAAACGAT), but with with slight modifications than in later selection rounds. Seven clones show one over the entire randomized area tense motif 1/1 that only three successive positions allows any variability. It contains that in the center Motif 1. (ggacGACATGTTC NNN GAACATACGGTGAAAGAAACGATTGTCGgacagg). In addition, two clones reappear in the sequencing of STAR-R02-12MW. Another can be found in STAR-R02-15d.

Selection with higher stringency

Selection round 6 was repeated, with several parallel approaches being selected. These differed only in a falling peptide concentration. In the following rounds, the peptide concentration was reduced further and further. The peptide concentrations were selected as follows: The highest peptide concentration corresponded to the concentration at which the previous round had still been successfully selected (signal> 1%, at least 3 times via empty selection). In addition, peptide concentrations reduced by a factor of 3.16 and a factor of 10 were used. An empty selection without any peptide served as a control. Rounds 11-14 were selected with a reduced RNA: peptide ratio of 5: 1, round 15 as a double round (2x selection with no amplification). The result is shown graphically in FIG. 30 and in a table in FIG. 31.

Sequencing of selection round STAR-R02-15d

22 different sequences were obtained from 41 evaluable sequences, all of which characteristic motif 1 (CATACGGTGAAAGAAACGAT). 14 of them showed the expanded motif 1/1. 20 of the 41 clones also appear in selection STAR-RNA-03-11 parallel 2-step ligation. 17 partially overlapping can be found in STAR-R02- 12 MW again, 3 in the 37 ° C selection STAR-R02-15xx. A single sequence can be found in Selection STAR-R02-10.

matrix changing

From round 8, another parallel selection strand was opened based on round 75 (1 µM). Compared to the previously mentioned selection strands, this is characterized by the repeated change of the selection matrix (solid phase). It was switched between streptavidin ultra-link Plus (Pierce / Perbio) and neutravidin-agarose (Pierce / Perbio). The result is shown graphically in FIG. 32 and in a table in FIG. 33.

Sequencing of selection round STAR-R02-12MW

From 35 evaluable sequences, 22 different sequences were obtained (1 of which has an N). 20 of the 22 (19 without N of the 22) sequences carry the characteristic motif 1, the was also observed in selection STAR-R02-15d (CATACGGTGAAAGAAACGAT). 11 clones consist of the expanded motif 1/1. 2 of the 35 clones are known from selection STAR-R02-10, 21 from STAR-R02-15d. 2 also come in STAR-R02-15xx and 15 clones in STAR-R03-11.

Selection at 37 ° C

From round 10, a further strand was branched off from the matrix change selection (RNA 02MW) and further selected at 37 ° C. Rounds 13xx, 14xx, 15xx were selected as double rounds. The result is shown graphically in FIG. 34 and in a table in FIG. 35.

Sequencing of selection round STAR-R02-15xx

Cloning and sequencing provided 40 evaluable clones with 17 different ones Sequences (3 of them have an N). 6 sequences (5 without a seq. With N) carry the typical Motif 1 (CATACGGTGAAAGAAACGAT). 4 (3 without N) sequences have a partial motif from the above: (GAAAG) and are almost identical. A clone shows that characteristic motif 1/1. 7 clones (6 of which are identical) carry the 1/1 motif with four successive variable positions (ggacGACATGTTC NNNN GAACATACGGTGAAAGAAACGATTGTCGgacagg).

This selection strand gave rise to clone R02-15xx-A11, which was used in Examples 9 and 11 was characterized in the cell culture and in Example 12 in the micro-calorimeter.

6 (5 without one with N) sequences carry a newly identified one with slight variations Motive 2 (GATGGCGCGGTCTNAAAAAACGCCGNNNGGGNGAGGG). Another one very similar with a variation (6 nt) in the center of the motif. Of the 40 clones there were already three identical ones in selection STAR-R02-10. Two entered STAR R02-15d on. One of them also in STAR-R02-12MW. 7 clones were in selection R03-11 Find.

Parallel 2-step ligation: selection STAR-R03

The selection, the eluates of which were first divided and then only 5 'and then 3' or first 3 'and then 5', was carried out with increasing stringency until round 11. The result is shown graphically in FIG. 36 and in a table in FIG. 37.

Sequencing of selection round STAR-R03-11

Cloning and sequencing provided 29 different sequences from 47 clones. 26 of which contained the characteristic motif (CATACGGTGAAAGAAACGAT). one The sequence lacks the 5 ° C (→ A). A sequence shows the shortened motif (GAAAG, see STAR-R02-15xx). 14 of the 26 showed the expanded motif 1/1. Two more the motif 1/1 with four successive variable nucleotides, which is also in selection STAR-R02- 15xx occurred. A sequence is obviously an orphan.

There is no sequence overlap with STAR-R02-10. 20 clones were also found in Selection STAR-R02-15d, 19 clones in selection STAR-R02-12MW and 7 clones in STAR- R02-15xx.

Overall, there is no difference at the sequence level due to the large overlap between simultaneous and parallel 2-step ligation. From two clones Modified molecules have already been synthesized (shortened and terminal stem ) Stabilized. These are: STAR-R03-11-F10-45-001 and STAR-R03-11-C12-48-001.

From this selection the clone R03-11-F10 emerged, which in Example 12 in Microcalorimeter was characterized in more detail.

The various sequences obtained are shown in Figs. 43-47.

Example 5 Selection of an aptamer binding to biotinylated CGRP under Use of unmodified RNA

This example describes the selection of aptamers that bind to rat CGRP, more precisely biotinylated CGRP. The CGRP was acquired by Jerini AG and has 37 amino acids with a pI according to SWISS PROT database entry of 7.8. Neutravidin-agarose was used as the matrix; after round 9, a switch was made to magnetic streptavidin beads corresponding to the materials used in Example 3. The immobilization for the selection with pre-immobilized CGRP was carried out on NeutrAvidin agarose using the following buffer, which was also used as a selection buffer: 10 mM HEPES (pH 7.4), 1 mM CaCl ₂ , 1 mM MgCl ₂ , 4 mM KCl, 150mM NaCl, 0.1% Tween 20.

The complex formation took place on the matrix, after round 9 in solution. The elution took place by affinity elution for the pre-immobilized target, with 8 M urea for the Streptavidin beads. The selection temperature was 37 ° C and the pool was the RNA pool used as used in connection with Example 1, except that no 2'-fluorine-modified pyrimidine nucleotides were used here.

Characteristics of the selection

The selections were made at a constant concentration of the target molecule from 50 µM to for round 9. From round 9, this concentration gradually increased to 10 nM lowered to slowly increase the stringency of the selection. In the course of the selection starting from a 2.5-fold wash volume, the wash share was 5-fold (Laps 3-6) or 10-fold (from lap 7) wash volume increased. The target molecule was in Rounds 1-8 pre-immobilized on NeutrAvidin agarose. In the following rounds (from Round 9) the complex was formed in solution on magnetic streptavidin beads. As Elution method was eluted for 5 minutes with 7 M urea at 65 ° C.

Selection process

During the course of the selection, a first increase in round 4 was observed. However, this increase was only successful in round 9 with a 17% connection. Since an increase in the precolumn was also observed in this round, the switch to magnetic streptavidin beads mentioned above took place in this round, with about 10% of the RNA still showing a link. By increasing the stringency of the selection, attempts were made to improve the quality of the binding molecules with regard to a higher binding constant. In round 15, a binding rate of 16% was achieved at a concentration of 500 nM of the target molecule and the pool was cloned and sequenced. The sequencing resulted in 4 sequence families, which were strongly represented with 14 to 32 clones, a further 13 sequences were present 2 to 7 times in the pool. 36 individual sequences were also found. The determination of binding constants using the Biacore device revealed affinities that were still too far from the target affinity of K _D <30 nM. The best binding constant was determined for the single sequence G12. Based on this result, the selection was continued with further increase in stringency until round 19 (concentration of target molecule 10 nM). In round 18, a connection was carried out as a control experiment with a concentration of the target molecule of 5 μM and a ratio of RNA to target molecule of 1: 1, a 50% binding being achieved. After completing round 19 at 10 nM and a binding rate of 8.5%, the pool obtained was sequenced. A sequence family could be identified from the result of the sequencing, which makes up over 60% of the pool. This sequence family corresponded to clone G12, the best binder from the sequencing after round 15 (where G12 was a single sequence there). A K _D of 100-200 nM at 25 ° C. was determined for the clone G12.

The results of the Biacore experiments suggest a bivalent binding behavior of the clone. This could be shown in experiments with non-biotinylated CGRP. Also shows the Repeat round 19 with affinity elution, in which the elution of the biotinylated CGRP-bound RNA with free, non-biotinylated CGRP occurs only a small amount Proportion of elutable RNA. This round 19 with affinity elution was not sequenced. Furthermore, in the sequencing of round 19 with denaturing elution next to the dominant G12 clone another motif family with 14 representatives and 30 new ones Individual sequences found. Determining the dissociation constant using the Biacore device showed that all of these new sequences have lower affinity and thus had a worse dissociation constant than clone G12.

The course of the selection is shown in the table below.

The preferred individual sequences can be represented as follows.

G12 - group 12

+ Mutations

the K _D values varied only in a very narrow band. H08 - Group 6

The binding nucleic acids of this group show K _D values worse than the nucleic acid according to clone G 12. H03 - group 6

The binding nucleic acids of this group show a K _D value worse than the nucleic acid according to clone G12.

Example 6 Synthesis of L-2'-fluoro-phosphoramidites

The synthesis of 5'-DMT-2'-fluoro-L-uridine and 5'-DMT-2'-fluoro-N (Ac) -L-cytidine phosphoramidites (3 and 5) is shown in Figs. 15 to 17 illustrates:
2'-Fluoro-L-uridine 2 was synthesized from L-arabinose 1 according to Codington, JF, Doerr, IL, & Fox, JJ (1964) J. Org. Chem. 29, 558. It was then converted to 5'-DMT-2'-fluoro-L-uridine and phosphitylated ( Fig. 15) to obtain the phosphoramidite ( Fig. 17).

The references according to FIG. 15 mean:
a) cyanamide, MeOH, NH ₄ OH; b) methyl propiolate, EtOHaq; c) HF, dioxane, autoclave 120 ° C; d) DMTCl, DMAP, Pyr; e) Cl-P (OCH ₂ CH ₂ CN) N (iPr) ₂ , DIPEA, DMAP, THF.

For the conversion of D-uridine derivatives into D-cytidine derivatives, the literature (Sung WL (1982) J. Org. Chem. 47, 3623; Reese CB, Ubasawa A. (1980) Tetrahedron Lett., 2265; Sung WL (1981) J. Chem. Soc. B, 1089; Krug A., Schmidt S., Lekschas J., Lemke K., Cech D. (1989) Journal f. Practical Chemistry, 331 (5), 835 ) described many synthetic routes. These syntheses use 1,2,4-triazole (Sung WL (1982) J. Org. Chem. 47, 3623) or 1-tetrazole (Reese CB, Ubasawa A. (1980) Tetrahedron Lett., 2265; Sung WL (1981) J. Chem, Soc. B, 1089; Krug A., Schmidt S., Lekschas J., Lemke K., Cech D. (1989) Journal f. Practical Chemistry, 331 (5), 835) , The synthesis of 2'-fluoro-L-cytidine 4 was carried out using 1,2,4-triazole chemistry (Krug A., Schmidt S., Lekschas J., Lemke K., Cech D. (1989) Journal f. Prakt. Chemie, 331 (5), 835). The 2'-fluoro-L-cytidine 4 thus obtained was then aminoacetylated, tritylated and phosphorylated ( FIG. 16) in order to obtain 5.

The references according to FIG. 16 mean:
a) BzCl, Pyr; b) 1,2,4-triazole, 4-chlorophenylphosphorodichloridate, pyr; c) NH ₃ (28% in water) / dioxane; d) Ac ₂ O, DMF; e) DMTCI, DMAP, Pyr; f) Cl-P (OCH ₂ CH ₂ CN) N (iPr) ₂ , DIPEA, DMAP, THF.

The details of the conversion of 2'-fluoro-L-uridine 2 to 2'-fluoro-L-cytidine 4 are described in the following figure ( Fig. 17):
The references according to FIG. 17 mean:
a) BzCl, Pyr; 1b) 1,2,4-triazole, 4-chlorophenylphosphorodichloridate, pyr; c) NH ₃ (28% in water) / dioxane; or NH ₃ (28% in water) / dioxane and MeO / NH ₃ sat.

2'-Fluoro-L-uridine 2 was first benzoylated to give 6. Substance 6 was without Purification continues directly with 1,2,4-triazole and 4-chlorophenylphosphorodichloridate implemented to give 7. After treatment with ammonia / dioxane, 2'-fluoro-L- cytidine directly from the unpurified substance as a beige-brown crystallization product be preserved.

Of particular interest with this type of synthesis is that no purification of the Intermediate substances is necessary. The final substance can differ from the raw substance with high Yield of about 70% can be obtained.

experimental setup

2'-Fluoro-L-uridine 2 (22.35 g, 57% purity, 90 mmol) was coevaporated three times with dry pyridine (3 × 40 ml). The crude substance was introduced into dry pyridine (150 ml, with molecular sieve) and cooled to 0 ° C. Benzoyl chloride was added dropwise and the reaction was stirred for 1 h at 0 ° C and stored overnight at room temperature (TLC (Hex / EE 1/2): Rf: 0.5). The reaction was quenched with methanol (5 ml) and concentrated to dryness. The residual substance was dissolved in DCM (250 ml) and diluted with NaHCO ₃ (50%, 150 ml, three times). The organic phase was dried over Na ₂ SO ₄ and concentrated.

The crude substance was dissolved in dry pyridine (300 ml). The mixture was stirred with molecular sieve at 0 ° C under N _{2 for} 30 minutes. 1,2,4-triazole (4.0 eq, 360 mmol, 29.9 g) was then added. This mixture was stirred at room temperature for 10 minutes. Then 4-chlorophenyl phosphodichloridate (2.0 eq, 180 mmol, 29.3 ml) was slowly added to the mixture. After 30 minutes the reaction became exothermic.

The mixture was stirred at 0 ° C for 2 h and stored at room temperature overnight. The unpurified product ran in a spot on the thin layer plate (TLC, hexane / ethyl acetate 1/2): Rf: 0.34). The residual substance was dissolved in DCM (250 ml) and the organic phase was washed with dilute NaHCO ₃ (50%, 300 ml) (exothermic). The organic phases were dried over Na ₂ SO ₄ and evaporated.

The crude substance was dissolved in dioxane (600 ml) and NH ₃ (600 ml, 28% in water), then stirred for 72 h at room temperature. After the TLC control (DCM / MeOH 9/1, Rf: 0.34), the mixture was evaporated to dryness (brownish oil) and coevaporated with methanol (twice 50 ml). The brownish residue was dissolved in a mixture of water (200 ml) and DCM (200 ml). The aqueous phase was then washed three times with DCM (150 ml) and concentrated until a minimal volume (50 ml) was reached. After a night in the refrigerator, the substance was filtered, washed with DCM (twice 25 ml) and Et ₂ O (2 × 25 ml) (beige-brown crystals, 6.1 g (50% yield). A second crop (3, 0 g, 23% yield) was obtained after a short chromatography of the original substance (storage on silica gel, course of MeOH in DCM (5-30%)) and a second crystallization from water (total yield 73%).

An alternative method could result from the suspension of the raw substance in dioxane (300 ml) and NH ₃ (300 ml, 28% in water). The mixture was stirred at room temperature for 2 hours. After the TLC control (DCM / MeOH 9/1) the mixture was evaporated to dryness (brownish oil) and coevaporated with methanol (twice 50 ml). The crude substance was dissolved in methanol (approx. 5 ml) and saturated methanolic ammonia (200 ml) was added. The mixture was stirred overnight.

Example 7 Determination of the cytotoxicity of selected LGR binding to CGRP nucleic acids

On the basis of the L-nucleic acids selected in Example 2, one of the Compounds, namely 732_096, are used to reduce the toxicity of L-nucleic acid determine. The neuroblastoma cell line SK-N-MC was used as a cellular test system Accession number ACC 203 (DSMZ, German Collection of Microorganisms and Zellkulturen GmbH, Braunschweig) used

AlamarBlue test

The AlamarBlue Test is an assay for the detection of cell growth and cytotoxicity. AlamarBlue is an indicator dye for the quantitative measurement of the proliferation of eukaryotic and prokaryotic cells. AlamarBlue is a non-toxic, water-soluble Oxidation-reduction indicator which, when chemical reduction has taken place, is both effective Fluorescence as well as its colorimetric properties changes. Chemical reduction of Indicator is found in living cells proportional to cellular metabolism and intracellular enzyme activity takes place and is therefore a measure of the cytotoxicity tested Substances (Ahmend et al., 1994, J. Immunol. Methods 170, 211-224, Page et al., 1993, Int. J. Oncology 3, 473-476 ,. The absorption can be done with a microtiter plate photometer 570 nm (excitation) (600 nm emission; reference) can be measured.

To do this, AlamarBlue is diluted 5% in culture medium, one with 100 µl / well Microtiter plate (96 format) on the previously with the test substances (Spiegelmere) 30 Minutes of incubated cells and incubated for a certain time in the incubator.

The incubation period depends on the type and number of cells. As a guide, apply to the used SK-N-MC cells (neuroblastoma cell line) 2 h. At the end of the incubation period the photometric measurement takes place.

The percentage change in the treated wells is used as a measure of the cytotoxicity the respective controls determined. Inhibitions greater than 20% are considered cytotoxic effects considered.

The result is tabulated in the table below.

Example 8 Inhibition of cAMP production by human CGRP binding Spiegelmers®

The analysis of the biological effectiveness of CGRP-binding L-nucleic acids, i. H. of Spiegelmeren was done as follows.

Cells of the human neuroblastoma line SK-N-MC with the accession number ACC 203 (DSMZ, German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig) are sown in a number of 4 × 10 ⁴ per well of a 96 well microtiter plate and at 37 ° C and 5% CO ₂ in DMEM (1,000 mg / L glucose), which additionally contains 10% heat-inactivated fetal calf serum (FCS), 4 mM L-alanyl-L-glutamine (GLUTAMAX), 50 units / ml penicillin, 50 µg / ml contains streptomycin, cultured. 48 hours after sowing, the cells are 80-90% confluent and are used for the experiments.

The Spiegelmers are combined with 1 nM human or rat CGRP (Bachem) Hank's balanced salt solution (HBSS) + 1 mg / ml BSA for 15-60 min at RT or 37 ° C in incubated a 0.2 ml "low profile 96-tube" plate. Just before being added to the cells 2 ul of a 50 mM IBMX solution added. The cells are 20 min before the addition of CGRP / Spiegelmer approaches pretreated with 1 mM IBMX.

For stimulation, the medium is aspirated from the cells and the pre-incubated batches are added. After incubation for 30 min at 37 ° C., the cell supernatants are suctioned off and the cells are lysed with 50 μl / well lysis buffer for 30 min at 37 ° C. The lysis buffer is part of the "cAMP-Screen ™ System" kit (Applied Biosystems), with which the cAMP content of the extracts is determined. 10 µl of the extracts are used in the test. The test is carried out as described by the manufacturer:
In an assay plate (coated with goat anti-rabbit IgG), 10 ul / well of the lysate are added to 50 ul of the lysis buffer and mixed with 30 ul / well of the cAMP-alkaline phosphatase conjugate diluted according to the manufacturer's instructions. Then 60 µl / well of the cAMP antibody included in the kit is added. This is followed by an incubation period of 1 h with shaking at room temperature. The solutions are then removed from the wells and washed six times with the washing buffer provided. For detection, 100 μl / well CSPD / Sapphire-II RTU substrate are added, incubated at RT for 30 min and the luminescence is measured in a POLARstar Galaxy multi-detection plate reader (BMG).

In this test system, two L- described in Example 2 herein Nucleic acids tested. These are the sequences 732_096 and 732_045 according to the SEQ. ID. No 14 and 13.

The Spiegelmers 732_045 and 732_096 were added together with the cells Pre-incubate 1 nM human CGRP in medium (M199) without serum for 15 min. After 30 min the cells were lysed at 37 ° C. The extracts from two treated equally "Wells" were combined and the amounts of cAMP determined as described.

The result is shown in Fig. 23. The amounts of cAMP formed were applied as a percentage of the control. The graphic evaluation shows the concentration of Spiegelmer, at which only 50% of the amount of cAMP present in the control is formed (IC50), a value of approx. 60 nM for 732_096 (59mer) and approx. 22 nM for 732_045 ( 55mer). These very good IC50 values, which also correlate with the dissociation constants, clearly show that the identified fluoro-Spiegelmers are extremely potent, more potent than the unmodified RNA molecules previously selected with the same methods.

Example 9 Inhibition of cAMP production by rat CGRP binding Spiegelmers®

The test was carried out as described in connection with Example 8. The Spiegelmer L 501L, which had been selected in the context of Example 4, was preincubated together with 1 nM rat CGRP in Hank's balanced salt solution (HBSS) + 1 mg / ml BSA for 60 min at 37 ° C. before addition to the cells , After 30 minutes of stimulation, the cells were lysed and the amounts of cAMP in the lysates were determined as described. Triplicate determinations were carried out and the amounts of cAMP formed as a percentage of the control (no Spiegelmer in the preincubation approach) ± standard deviation plotted in FIG. 24. The graphic evaluation shows the concentration of Spiegelmer in which only 50% of the cAMP present in the control Amount is formed, a value of approximately 60 nM.

With a half-maximum stimulating concentration by CGRP of approximately 1 nM, as determined in Example 11, this results in a K _i value of approximately 30 nM.

Example 10 Binding studies on cellular CGRP receptor

Membranes of CHO-K1 cells which are transfected with human calcitonin-receptor related receptor (CRLR, Genbank: U17473) and human receptor-associated modifying protein type 1 (RAMP1, Genbank: AJ001014) (Fa. Euroscreen, Brussels, Belgium), are thawed rapidly, diluted in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl ₂ , 1 mM EDTA pH 7.4 and resuspended by homogenization. For each test batch, 2 µg membrane protein in 20 mM HEPES pH 7.4, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM EDTA, 1 µM GDP and the Spiegelmere are incubated in a volume of 150 µl. After 30 minutes of incubation at room temperature, 50 μl of 2 nM [ ³⁵ S] GTPγS (from Amersham) are added and incubated for a further 30 minutes at room temperature. Then 25 μg of WGA-PVT beads (wheat germ agglutinin-loaded polyethyleneimine-treated) (Amersham) are pipetted into 50 μl, incubated for 30 minutes at room temperature and the assay plate is centrifuged for 10 min at 3200 rpm. Bound activity was determined using a Wallac1450 MicroBeta ™ scintillation counter (Wallac).

Evaluation. The data are analyzed by non-linear regression (GraphPad Prism, 3.02, Graphpad Software Inc., San Diego, CA, USA).

The binding studies were carried out with the two Spiegelmeres 732_045 and 732_096, which were selected in Example 2. The result is shown in Fig. 25.

Both Spiegelmers efficiently inhibit the binding of the ligand to the receptor. This means that the Spiegelmer specifically recognizes the CGRP, captures it and therefore there is no receptor binding and receptor activation. As a result, no [ ³⁵ S] GTPγS can be incorporated.

732_045 and 732_096 were each cleaned using HPLC or gel. FIG. 25 shows that the purification method within the scope of the error has only an insignificant influence on the biological activity of the fluoro-modified RNA mirror mers.

Example 11 Cell culture experiments - dose-response curve for CGRP

SK-N-MC cells were cultivated in 96-well plates as described in Example 8. 20 min before stimulation with rats α-CGRP, the medium was replaced with 100 μl Hank's balanced salt solution (HBSS) + 1 mg / ml BSA and 1 mM 3-isobutyl-1-methylxanthine (IBMX) was added. The stimulation with CGRP was carried out by adding 100 or 30 times concentrated stock solutions of rat α-L-CGRP (Bachem) dissolved in PBS. After 30 min at 37 ° C., the supernatant was suctioned off and the cells were lysed with 50 μl lysis buffer. The cAMP content of the extracts was determined as described in Example 8. The plotting of the amounts of cAMP formed against the CGRP concentration used for the stimulation, as shown in FIG. 38, resulted in a half-maximal activation of approximately 1 nM.

Example 12 Calorimetric determination of binding constants and activity execution

The binding of Spiegelmeres to rats alpha-CGRP was also determined using the method of Isothermal titration calorimetry (ITC) determined with the VP ITC (Microcal).

For this purpose, 1.465 ml of a 10 µM solution of the Spiegelmers were placed in the adiabatic measuring cell of the Device submitted. Free when adding 7.5 µl of a 70 µM rat α-L-CGRP solution enthalpy of binding is registered by the device. By adding the Peptides and measurement of the binding enthalpies released in each case Binding constants and activities of the molecules can be determined.

The Spiegelmer STAR-R02-15xx-A11 was measured at 37 ° C. The result is in the Figs. 39 and 40 are shown.

The Spiegelmer STAR-R03-F10 was measured at 25 ° C. The result is shown in Figs. 41 and 42.

Results of the ITC measurement

The binding constant or dissociation constant K _D is the reciprocal of the association constant K _A (K in the enthalpy diagram) and was found to be 65 nM for the Spiegelmer STAR-R02-15xx-A11. The activity was 42%. This is slightly worse than cell culture data, but of the same order of magnitude.

Spiegelmer STAR-R03-11-F10 showed a binding constant of 28 nM and one at 25 ° C Activity of 83%.

The features of the invention disclosed in the preceding description, the claims and the drawings can be essential both individually and in any combination for realizing the invention in its various embodiments. SEQUENCE LISTING

Claims (33)

1. An antagonist of CGRP, the antagonist being a nucleic acid and preferably the nucleic acid binds to CGRP. 2. The antagonist of CGRP according to claim 1, wherein the CGRP is α-CGRP. 3. The antagonist according to claim 1, characterized in that the CGRP β-CGRP is. 4. An antagonist of the CGRP1 receptor, the antagonist being a nucleic acid and
wherein preferably the nucleic acid binds to a ligand of the receptor and
preferably the ligand is CGRP. 5. The antagonist according to claim 4, characterized in that the ligand α-CGRP is. 6. The antagonist according to claim 4, characterized in that the ligand is β-CGRP is. 7. The antagonist according to one of claims 1 to 6, characterized in that the Nucleic acid comprises at least one L nucleotide. 8. The antagonist according to claim 7, characterized in that the antagonist is an L- Is nucleic acid. 9. A nucleic acid that binds to CGRP. 10. The nucleic acid according to claim 9, characterized in that the CGRP α-CGRP is. 11. The nucleic acid according to claim 9, characterized in that the CGRP β-CGRP is. 12. A nucleic acid with a sequence, the sequence being selected from the Group comprising the sequences according to SEQ ID No. 1 to SEQ ID. No. 244th 13. The nucleic acid according to any one of claims 9 to 12, characterized in that the nucleic acid comprises at least one L nucleotide. 14. The nucleic acid according to any one of claims 9 to 13, characterized in that the nucleic acid is an L-nucleic acid. 15. The nucleic acid according to any one of claims 9 to 14, characterized in that the nucleic acid is selected from the group comprising DNA, RNA and Combinations of these. 16. The nucleic acid according to any one of claims 9 to 15, characterized in that the affinity of the nucleic acid is less than 0.5 µM, preferably less than 0.1 µM, preferably less than 0.05 µM and most preferably less than 0.01 µM is. 17. The nucleic acid according to any one of claims 9 to 16, characterized in that the affinity of the nucleic acid more than 250 nM, preferably more than 100 nM, more preferably is more than 50 nM and most preferably more than 10 nM. 18. The nucleic acid according to any one of claims 9 to 17, characterized in that the nucleic acid comprises a minimal binding motif. 19. The nucleic acid according to any one of claims 9 to 18, characterized in that it has a length, the length being selected from the group, the lengths from 15 to 150 nucleotides, 20 to 120 nucleotides, 30 to 120 nucleotides, 40 to 100 nucleotides, 40 to 70 nucleotides and 50 to 70 nucleotides, 30 to 50 and am most preferably comprises 25 to 45 nucleotides. 20. The nucleic acid according to any one of claims 9 to 19, characterized in that the nucleic acid has a two, three or more part structure. 21. Use of a nucleic acid according to one of claims 9 to 20 as an antagonist of CGRP and / or the CGRP1 receptor system. 22. Use of a nucleic acid according to one of claims 9 to 20 for the Manufacture of a drug. 23. Use of an antagonist according to one of claims 1 to 8 for the preparation of a drug. 24. Use according to claim 22 or 23, characterized in that the Drug for the treatment of a disease is selected from the group is, the migraine, cluster headache, loss of appetite, nausea, vomiting, neurogenic inflammation, especially neurogenic inflammation that is shared with others Neuropeptides is mediated, vasodilation, increased blood pressure, hypotension, Tachicadia, disorders related to trigeminal afferent activation sensory neurons and central "nociceptive" neurons, especially higher ones Pain centers, decrease, and chronic inflammatory pain, includes and / or for the treatment of pain, especially chronic pain, acute Pain, inflammatory pain, visceral pain and neuropathic Pain. 25. A composition comprising a nucleic acid according to any one of claims 9 to 20 and preferably a pharmaceutically acceptable carrier. 26. A composition comprising an antagonist according to one of the preceding Claims and preferably a pharmaceutically acceptable carrier. 27. Complex comprising CGRP and a nucleic acid according to any one of claims 9 to 20th 28. Use of a nucleic acid according to one of claims 9 to 20 for detection of CGRP, preferably α-CGRP or β-CGRP and most preferred human α-CGRP or β-CGRP. 29. A method for screening CGRP antagonists comprising the following steps: Providing a candidate CGRP antagonist, Provision of a nucleic acid according to one of claims 9 to 20, Provision of a test system which gives a signal in the presence of a CGRP antagonist, and - Determine whether the candidate CGRP antagonist is a CGRP antagonist. 30. The method according to claim 29, wherein the CGRP is α-CGRP and / or β-CGRP, preferably human α-CGRP and / or β-CGRP. 31. A method for screening CGRP agonists comprising the following steps: - providing CGRP, Provision of a nucleic acid according to one of claims 9 to 20, preferably a labeled nucleic acid according to one of claims 9 to 20, - addition of a candidate CGRP agonsite, and - Determine whether the candidate CGRP agonist is a CGRP agonist. 32. The method according to claim 31, characterized in that the determining thereby it is determined that the nucleic acid is found by the candidate CGRP agonist is ousted. 33. Kit for the detection of CGRP, preferably α-CGRP or β-CGRP, comprising a nucleic acid according to any one of claims 9 to 20.