Antisense Oligonucleotides Against VR1
Antisense oligodeoxynucleotides against VR1, corresponding nucleotide constructs, cells containing said nucleotide constructs, pharmaceutical and diagnostic substances, uses thereof in pain therapy, and methods for diagnosing symptoms related to VR1 and for identifying pain-modulating substances.
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The present application is a division of application Ser. No. 10/376,341, filed Mar. 3, 2003, now U.S. patent Ser. No. ______, which in turn is a continuation of International Patent Application No. PCT/EP01/10081, filed Aug. 31, 2001, designating the United States of America and published in German as WO 02/18407 A2, the entire disclosure of which is incorporated herein by reference. Priority is claimed based on Federal Republic of Germany Patent Application Nos. 100 43 674.9, filed Sep. 2, 2000, and 100 43 702.8, filed Sep. 4, 2000.
BACKGROUND AND SUMMARY OF THE INVENTIONThe invention relates to antisense oligodeoxynucleotides against VR1, to corresponding nucleotide constructs, to cells, pharmaceutical preparations and diagnostic preparations containing these, to the use thereof in pain therapy and to methods for diagnosing symptoms associated with VR1 and for identifying pain-modulating substances.
The effective treatment of pain is a major challenge to molecular medicine. Acute and transient pain is an important bodily signal protecting people from severe injury by their environment or by overloading their body. In contrast, chronic pain, which lasts longer than the cause of the pain and the anticipated time frame for cure, has no known biological function and affects hundreds of millions of people worldwide. In the Federal Republic of Germany alone, some 7.5 million people suffer from chronic pain. Unfortunately, pharmacological treatment of chronic pain is still unsatisfactory and thus remains a challenge to current medical research. Currently existing analgesics often have an inadequate action and sometimes have severe side effects.
The search is thus now on for new targets or endogenous body structures which appear to provide a way of exerting pain-modulating action, for example using low molecular weight active substances or other compounds such as antisense oligodeoxynucleotides (ODN), in particular for the treatment of chronic pain.
The vanilloid receptor subtype 1 (VR1, also known as the capsaicin receptor) cloned by Caterina et al. (1997) is a promising starting point for the development of new analgesic drugs. This receptor is a cation channel which is predominantly expressed by primary sensory neurons (Catarina et al. 1997). VR1 is activated by capsaicin, a component of chillies, heat (>43° C.) and a low pH as a result of tissue injury, and brings about a calcium influx in primary afferents. VR1 knockout mice did not develop thermal hyperalgesia after tissue injury or inflammation (Caterina et al., 2000; Davis et al., 2000).
Antisense oligodeoxynucleotides, ribozymes and other catalytic nucleic acids may be used for the treatment, in particular of chronic pain, by degrading or modifying the mRNA of selected targets, in the case of the present invention the above-described VR1, to down-regulate the expression thereof and thus reduce the number of receptors per cell. The ODN attach themselves to the mRNA, so firstly blocking translation and secondly initiating degradation of the mRNA by RNase H, which cleaves the DNA/RNA duplex. Porreca et al. (1999) were able to demonstrate that intrathecally administered ODNs against the PN3/SNS channel in rats prevent the development of hyperalgesia and allodynia due to chronic nerve or tissue damage.
Even once the sequence of VR1 is known, effective blocking and cleavage of the mRNA in particular depends upon the selection of the correct antisense oligodeoxynucleotides, ribozymes and other catalytic nucleic acids. The target's mRNA is usually folded and only a few sites are accessible for attachment and subsequent cleavage. Nothing is known in the prior art about how to select the ODN correctly.
DESCRIPTION OF THE INVENTIONThe object of the present document was accordingly to develop antisense oligodeoxynucleotides and catalytic nucleic acids together with corresponding ribozymes against the mRNA of the vanilloid receptor. The present invention accordingly provides an oligonucleotide containing or corresponding to a base sequence according to one of sequences (b) to (j) in each of
The present invention also provides an oligonucleotide containing or corresponding to a base sequence according to one of sequences (b) to (j) in one of
The present invention also provides an oligonucleotide containing or corresponding to a base sequence according to (k) in one of
The present invention also provides an oligonucleotide containing or corresponding to a base sequence according to (k) in each of
The present invention preferably also provides an oligonucleotide which has a length of 15 to 30, preferably of 15 to 25, in particular 17 to 19, or exactly 18, nucleotides.
The present invention also preferably provides an oligonucleotide (hereinafter denoted oligonucleotide A) according to one of the above-stated oligonucleotide forms in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of
The present invention also preferably provides an oligonucleotide (hereinafter denoted oligonucleotide B) in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of
The present invention preferably provides an oligonucleotide according to the invention which comprises at least one modified or unmodified ribose, at least one modified or unmodified phosphodiester bond and/or at least one modified or unmodified base.
The present invention preferably provides an oligonucleotide according to the invention, in which at least one of the nucleotides, in particular two or more of the nucleotides, are “locked nucleic acids” (LNAs) or at least one of the nucleotides, in particular all of the nucleotides, are phosphorothioates, preferably one in which two or more of the nucleotides are “locked nucleic acids (LNAs). “Locked nucleic acids” (LNAs) are ribonucleotides which contain a methylene bridge which joins the 2′ oxygen of the ribose with the 4′ carbon (see
Preferred oligonucleotides are those in which the LNAs are located at the 5′ and 3′ end of the oligonucleotide, preferably in each case the final 2-5 nucleotides, in particular in each case the final 3 or 4 nucleotides, on the 3′ and 5′ end of the oligonucleotide are LNAs. Preferably, oligonucleotides of the invention have >6, in particular >8 contiguous nucleotides in the oligonucleotide that are not LNAs. Preferably, of the nucleotides shown in the sequence domain according to the particular, sequence (a) of the oligonucleotide according to one of
In the case of oligonucleotides modified with LNAs or phosphorothioates, it is particularly preferred if the oligonucleotide is an oligonucleotide A according to the invention or an oligonucleotide B according to the invention, preferably an oligonucleotide B according to the invention (see above).
In general, the present invention specifically provides nucleic acids, in particular oligonucleotides, in which two or more of the nucleotides are “locked nucleic acids” (LNAs), in which the LNAs are on the 5′ and 3′ end of the oligonucleotide, preferably in each case the final 2-5 nucleotides, in particular in each case the final 3 or 4 nucleotides, on the 3′ and 5′ end of the oligonucleotide are LNAs, and/or in which >6, in particular >8 contiguous nucleotides in the oligonucleotide are not LNAs. The embodiments previously explained with regard to the LNAs also apply to this subject matter of the invention.
The present invention also preferably provides a polynucleotide construct containing at least one oligonucleotide according to the invention. Polynucleotide construct should here be taken to have a very wide meaning. It includes RNA and DNA and nucleotides from a length of at least 20 nucleotides. A “recombinant” polynucleotide construct should here be taken to be a general designation for any kind of DNA or RNA molecules which have been obtained by in vitro linkage of DNA or RNA molecules. Polynucleotide is taken to have the following meaning: the underlying nucleotide is a fundamental nucleic acid building block essentially consisting of nucleic base, pentose and phosphoric acid. This corresponds to a high molecular weight polynucleotide prepared from two or more nucleotides linked together by phosphoric acid/pentose esterification. However, the invention also includes modified polynucleotides which, while they retain the sequence of bases, have a modified backbone instead of phosphoric acid/pentose.
The present invention also preferably provides a polynucleotide construct containing in two separate domains, two nucleotide subsequences Fragment I and Fragment III according to one of sequences (l)-(n) or the two nucleotide subsequences helix I and helix III according to one of sequences (o)-(q) in one of
The present invention also preferably provides a polynucleotide construct which codes for at least one oligonucleotide according to the invention. This in particular comprises DNA to be read out or a vector containing DNA or RNA, the product of which is or may be an oligonucleotide according to the invention.
The present invention also particularly preferably provides a polynucleotide construct according to the invention which comprises a ribozyme, a DNA enzyme, a vector, in particular an expression vector, or a peptide nucleic acid (PNA).
The following definitions apply for the purposes of the present invention:
-
- Cloning vector: A general name for nucleic acid molecules acting as vectors or carriers for foreign genes or parts thereof during cloning.
- Expression vector: A name for specially constructed cloning vectors which, once inserted in a suitable host cell, permit the transcription and translation of the foreign gene cloned into the vector.
- PNA: Standard international abbreviation for peptide nucleic acids. Peptide-linked amino acids here form a chain, the amino acids bearing as side chain a base capable of hybridisation with DNA or RNA.
- Sequence: A succession of nucleotides or amino acids. For the purposes of the present invention, sequence is intended to mean nucleic acid sequence.
- Ribozyme: A name for a catalytically active ribonucleic acid (for example ligase, endonuclease, polymerase, exonuclease), see for example hammerhead ribozyme according to
FIG. 24 or 25 and the description of the Figures or see Vaish, N. K. et al. (1998), Nucl. Acid Res. 26, 5237-5242. - DNA enzyme: A name for a DNA molecule which exhibits catalytic activity (for example ligase, endonuclease, polymerase, exonuclease), see for example DNA enzyme 10-23 according to
FIG. 26 and the description of the Figure, or see Santoro and Joyce (1997) Proc. Natl. Acad. Sci. USA 94, 4262-4266. - Catalytic RNA/DNA: A general name for ribozymes or DNA enzymes (see above).
The present invention also preferably provides a polynucleotide construct containing in two separate domains two nucleotide subsequences, as described above, wherein said construct comprises a ribozyme, preferably a “hammerhead” ribozyme, or a DNA enzyme, preferably a type 10-23 or 12-32 DNA enzyme. It is particularly preferred and selected if said construct comprises a DNA enzyme containing at least the nucleotide subsequences Fragment I and Fragment III according to one of sequences (l) to (n), preferably (n), in one of
It is particularly preferred and selected if said polynucleotide construct comprises a ribozyme containing at least the nucleotide subsequences helix I and helix III according to one of sequences (o) to (q), preferably (o), in one of FIGS. 1, 3, 4, 5, 6, 8, 11 or 12; preferably 1, 3 or 11, in particular 11; or preferably 4, 6, 8 or 12, in particular 4 or 12.
Specifically, a preferred embodiment may in particular be found in
Specifically, a preferred embodiment may in particular be found in
The type “10-23” DNA enzyme for sequence n according to
This DNA enzyme was named V15 (9/9), wherein the name indicates that the enzyme is oriented towards the GUC site of oligo V15 and the “recognition arms” in each case contain 9 nucleotides (Fragment I and III), for example according to Fragment I and Fragment III of sequence (n) in
The present invention also preferably provides a polynucleotide construct according to the invention, wherein it comprises at least one modified or unmodified ribose, at least one modified or unmodified phosphodiester bond and/or at least one modified or unmodified base.
The present invention also preferably provides an oligonucleotide according to the invention or polynucleotide construct according to the invention, wherein it is bound on a support, in particular a protein, preferably tet-, transportin or ferritin, and/or is packaged in a liposome.
The present invention also preferably provides a cell containing at least one oligonucleotide according to the invention and/or a polynucleotide construct according to the invention.
The present invention also preferably provides a pharmaceutical preparation containing at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention, optionally together with a suitable auxiliary substance and/or additive. The pharmaceutical preparations according to the invention may be administered in the form of solutions for injection, drops or juice, as semi-solid dosage forms in the form of granules, tablets, pellets, patches, capsules, dressings or aerosols and, in addition to the at least one subject matter of the invention, they also optionally contain, depending upon the pharmaceutical presentation, excipients, fillers, solvents, diluents, colorants and/or binders. Selection of the auxiliary substances and the quantities thereof which are to be used depends upon whether the pharmaceutical preparation is to be administered orally, perorally, parenterally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally, rectally or topically, for example onto infections of the skin, mucous membranes or eyes. Preparations in the form of tablets, coated tablets, capsules, granules, drops, succi and syrups are suitable for oral administration, while solutions, suspensions, easily reconstitutible dried preparations and sprays are suitable for parenteral, topical and inhalatory administration. Subject matters of the invention in a depot in dissolved form or in a dressing, optionally with the addition of skin penetration promoters, are suitable percutaneous administration preparations. Orally or percutaneously administrable formulations may release the subject matters of the invention in delayed manner. The quantity of active substance to be administered to the patient varies as a function of patient weight, mode of administration, the indication and the severity of the condition. 2 to 500 mg/kg of at least one subject matter of the invention are conventionally administered. Especially if the pharmaceutical preparation is to be used for gene therapy, suitable auxiliary substances and additives which it is advisable to use are, for example, a physiological saline solution, stabilizers, proteinase inhibitors, DNAse inhibitors etc.
The present invention also preferably provides a diagnostic aid containing at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention, optionally together with suitable additives.
The following definitions apply:
-
- Pharmaceutical preparation: A substance as defined in Article 1 §2 of the German Drug Law (Arzneimittelgesetz, AMG). In other words, substances or preparations made from substances which, by application on or in the human or animal body are intended
- 1. to cure, alleviate, prevent or diagnose diseases, suffering, bodily injury or sickness symptoms,
- 2. to diagnose the nature, the state or the functions of the body or mental health conditions,
- 3. to replace active substances or body fluids produced in the human or animal body,
- 4. to ward off pathogens, parasites or substances alien to the body or to destroy them or to render them harmless or
- 5. to influence either the nature, the state or the functions of the body or mental health conditions.
- Diagnostic aid: A compound or method which may be used to diagnose a disease.
The present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for the production of a pharmaceutical preparation for the treatment of pain, in particular chronic pain, tactile allodynia, thermally induced pain and/or inflammatory pain.
The present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for the production of a pharmaceutical preparation for the treatment of urinary incontinence; also of neurogenic bladder symptoms; pruritus, tumors, inflammation; in particular VR1 receptor-associated inflammation with symptoms such as asthma; together with any disease symptoms associated with VR1.
The present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for gene therapy, preferably in vivo or in vitro gene therapy. Gene therapy is taken to be a type of therapy in which, by the introduction of nucleic acids into cells, an effector gene product, usually a protein, but also an antisense oligodeoxynucleotide, is expressed. A fundamental distinction is drawn between in vivo and in vitro methods. In in vitro methods, cells are removed from the organism and transfected with vectors ex vivo before subsequently being introduced back into the same organism or a different one. In in vivo gene therapy, vectors, for example for combating tumors, are administered systemically (for example via the bloodstream) or directly into the tumor.
The present invention also preferably provides a process for the identification of pain-modulating substances, characterized in that identification is made by quantification of the binding of at least one, preferably labelled, oligonucleotide according to the invention or at least one polynucleotide construct onto an RNA.
The present invention also preferably provides a process for the identification of pain-modulating substances comprising the following process steps:
-
- (a) genetic engineering manipulation of at least one cell (test cell) with at least one oligonucleotide according to the invention and/or a polynucleotide construct according to the invention,
- (a′) parallel genetic engineering manipulation, of at least one identical cell (control cell), which manipulation is either
- not carried out,
- carried out in parallel with the oligonucleotide or polynucleotide construct, or
- carried out with a modified oligonucleotide or polynucleotide construct which is other than one according to the invention,
- (b) parallel incubation of a substance to be tested under suitable conditions with at least one test cell and at least one control cell and/or a preparation made from such a cell, which has synthesized at least one receptor protein selected from the vanilloid receptor family, preferably the VR-1 receptor,
- (c) measurement of the binding of the test substance onto the protein synthesized by the cells or measurement of at least one functional parameter modified by binding of the test substance onto the receptor protein,
- (d) identification of the substances by the extent of the difference between the measured value for the test cell and that for the control cell.
The term pain-modulating here refers to a potentially regulating influence upon physiological pain phenomena, in particular to an analgesic action. The term substance comprises any compound suitable as a pharmaceutical active substance, thus in particular low molecular weight active substances, but also others such as nucleic acids, fats, sugars, peptides or proteins such as antibodies. Incubation under suitable conditions should here be taken to mean that the substance to be investigated is capable of reacting in an aqueous medium for a defined period prior to measurement with the cell or the corresponding preparation. The aqueous medium may be temperature-controlled, for example between 4° C. and 40° C., preferably at room temperature or at 37° C. The incubation time may be varied between a few seconds and several hours, depending upon how the substance interacts with the receptor. Times of between 1 min and 60 min are, however, preferred. The aqueous medium may contain suitable salts and/or buffer systems, such that a pH of between 6 and 8, preferably pH 7.0-7.5 prevails in the medium during incubation. Suitable substances, such as coenzymes, nutrients etc., may additionally be added to the medium. Suitable conditions may readily be established by the person skilled in the art as a function of the substance-receptor interaction which is to be investigated on the basis of his/her experience, the literature or a few simple preliminary tests, so that the clearest possible measured value is obtained from the process. A cell which has synthesized a receptor is a cell which has already endogenously expressed this receptor or one which has been modified by genetic engineering in such a manner that it expresses this receptor and consequently contains the receptor prior to the beginning of the process according to the invention. The cells may be cells from possibly immortalized cell lines or be native cells originating and isolated from tissues, wherein cell aggregation has usually been broken down. The preparation made from these cells in particular comprises cell homogenates, the cytosol, a membrane fraction of the cells with membrane fragments, a suspension of isolated cell organelles etc.
The yardstick which permits the identification of interesting substances is either binding to the receptor, which may, for example, be detected by displacement of a known ligand or by the extent of substance binding, or the modification of a functional parameter due to the interaction of the substance with the receptor. This interaction may in particular involve regulation, inhibition and/or activation of receptors, ion channels and/or enzymes, while modified functional parameters may be, for example, gene expression, ion concentration, pH or membrane potential or modification of enzyme activity or the concentration of secondary messengers. The following definitions apply:
-
- subjected to genetic engineering manipulation: manipulation of cells, tissues or organisms in such a manner that genetic material is introduced or altered therein
- endogenously expressed: expression of a protein exhibited by a cell line under suitable culture conditions, without expression of this corresponding protein having been effected by genetic engineering manipulation.
Another preferred embodiment of the present process provides that the cell has already been subjected to genetic engineering manipulation before process steps (a) and (a′).
Another preferred embodiment of the present process provides that the genetic engineering manipulation permits the measurement of at least one of the functional parameters modified by the test substance.
Another preferred embodiment of the present process provides that the genetic engineering manipulation results in the expression of a form, not endogenously expressed in the cell, of a member of the vanilloid receptor family, preferably the VR-1 receptor, or in the introduction of a reporter gene.
Another preferred embodiment of the present process provides that binding is measured by the displacement of a known, labelled ligand of a member of the vanilloid receptor family, preferably the VR-1 receptor.
Another preferred embodiment of the present process provides that there is an elapse of not less than 8 h, preferably not less than 12 h, in particular not less than 24 h between the parallel process steps (a) and (a′), and process step (b).
The present invention also preferably provides a process for the diagnosis of clinical conditions which are associated with modified expression of genes of the vanilloid receptor family, which process is characterized in that the diagnosis is made by means of quantification of the binding of an oligonucleotide according to the invention and/or at least one polynucleotide construct onto an RNA.
The oligonucleotides and also the polynucleotide constructs are produced by processes known to the person ordinarily skilled in the art. Nucleotides, in particular also oligonucleotides, are synthesized in the manner of the Merrifield synthesis on an insoluble support (H. G. Gassen et al., Chemical and Enzymatic Synthesis of Gene Fragments (Verlag Chemie, Weinheim 1982)) or in another manner (Beyer/Walter; Lehrbuch der Organischen Chemie, 20th edition, (S. Hirzel Verlag, Stuttgart 1984), pp. 816 et seq.).
The present invention also provides a process for the treatment, in particular treatment for pain, of a non-human mammal or human requiring treatment for pain, in particular chronic pain, by administration of a pharmaceutical preparation according to the invention, in particular such a preparation containing an oligonucleotide according to the invention and/or a polynucleotide construct according to the invention. The invention also provides corresponding processes for the treatment of pruritus and/or urinary incontinence.
The following Examples and Figures are intended to illustrate the invention, but without restricting the subject matter of the invention thereto.
Figures, Figure and Figure should be regarded as synonymous. In relation to the Figures, the terms subtype and sequence are likewise synonymous. An “X” in the sequences shown denotes any desired complementary nucleotide to the corresponding base on the mRNA of VR1.
This DNA enzyme would be named V15 (9/9), wherein the name indicates that the enzyme is oriented towards the GUC site of oligo V15 and the “recognition arms” in each case contain 9 nucleotides (Fragment I and III), for example according to Fragment I and Fragment III of sequence (n) in
The first step in the antisense and ribozyme strategy is to identify accessible sites on the mRNA for binding oligonucleotides, in particular ribozymes. To this end, the VR1 mRNA had to be investigated for such restriction sites. Analysis of the VR1 mRNA revealed the following potential recognition sites for ribozymes in the coding domain:
33×GT(U)C sequences,
28×GT(U)T sequences, and
12×GT(U)A sequences.
In order to determine the accessible sites on the VR1 mRNA, in a first step, three independent nucleotide mixtures with the following sequence were synthesized:
-
- Mixture 1: NNNAACNNN “GUU library,”
- Mixture 2: NNNCACNNN “GUA library,” and
- Mixture 3: NNNGACNNN “GUC library.”
These were used consecutively in an RNase H experiment and it was found that appreciable degradation of the VR1 mRNA was observed only with the GUC library. Thus, of the potential target sequences for ribozymes, the 33 GUC sites in the VR1 mRNA are the most readily accessible and they were used for further analyses.
Example 2 Identification of the Most Effective Antisense OligodeoxynucleotidesMessenger Walk Screening
In order to identify mRNA domains which are accessible to antisense oligodeoxynucleotides, the mRNA was systematically screened with ODNs in an RNase H assay (messenger walk screening). The ODNs were 18 nucleotides in length and contained a central GAC sequence, which is reverse complementary to GUC sequences in the mRNA. This triplet was selected as the target as it provided good results and may be used in a second step to develop hammerhead ribozymes and DNA enzymes. In total, 33 ODNs, designated V1 to V33, were tested against all the GUC sites of the VR1 mRNA. The ODNs were systematically screened for their suitability by the addition in each case of one ODN and RNase H to the mRNA. RNase H cuts formed DNA/RNA duplexes wherever an oligonucleotide can bind to the mRNA (
In Vitro Transcription of VR1 mRNA
First of all, the cDNA of the vanilloid receptor was cloned into the vector pcDNA3.1 (+) from Invitrogen. Then, in vitro transcription of the mRNA was performed using the RiboMAX Large Scale RNA Production System—T7 from Promega in accordance with the manufacturer's instructions.
RNase H Assay
An RNase H assay was performed to test whether an antisense oligodeoxynucleotide had bound to the mRNA. To this end, the VR1 mRNA (100 mM) was incubated with a five-fold excess of the ODNs in a total volume of 10 μl in 40 mM tris/HCl pH 7.2, 4 mM MgCl2, 1 mM DTT and 150 mM NaCl for 7.5 minutes at 37° C. in the presence of 0.4 unit of RNase H (from Promega). The reactions were terminated by addition of EDTA (65 mM final concentration). The samples were separated on a 1.5% agarose gel and stained with ethidium bromide (1 μg/ml) for 20 minutes. The gels were photographed with the Gel Doc 2000 Gel Documentation System from Biorad and evaluated with Quantity One software.
Quantification of RNA degradation revealed that the most effective antisense oligonucleotide (oligo no. 30 (V30)) was able specifically to cut/cleave more than 90% of the VR1 mRNA when used in a five-fold excess relative to the target mRNA (
The intensities of the individual bands were evaluated.
The sequences of the antisense oligodeoxynucleotides most effective in this test are shown in
Oligodeoxynucleotides in which every fifth and six base (or in the event that these are identical, two adjacent bases) are swapped were synthesized and used in the following experiments as mismatch controls for these antisense oligodeoxynucleotides. The sequences of the control (mismatch) oligodeoxynucleotides are:
The RNase H assay was performed with the oligonucleotides V15, V15ctrl., V30 and V30ctrl. for control purposes (see
By using the RNase H assay with antisense oligodeoxynucleotides against all the GUC triplets of the VR1 mRNA, it was possible to identify oligodeoxynucleotides against the GUC triplets (2, 4, 15, 16, 26, 28, 30 and 32), in particular the 15th and 30th which were the ODNs with the best binding properties. These oligodeoxynucleotides and the two mismatch controls are thus available for testing in an animal model and for other uses.
The human sequences according to
On the basis of the identified most effective binding sites, corresponding ribozymes and DNA enzymes were also investigated.
“Hammerhead” ribozymes and type “10-23” DNA enzymes (Santoro et al., 1997) were constructed against the mRNA sites which were accessible to ODNs. The length of the “recognition arms” was 7 or 9 nucleotides on each side. Quantitative evaluation of mRNA cleavage/restriction under “single turnover” conditions (10-fold excess of ribozymes and DNA enzymes) revealed after 20 minutes at 37° C. that ribozymes with shorter “recognition arms” (helix I and helix III) are more active, while in turn in DNA enzymes, those with longer “arms” (Fragment I and Fragment III) are more active (
Experiments with ribozymes and DNA enzymes were carried out in 50 mM tris/HCl, pH 7.5 and 10 mM MgCl2 at 37° C. In the “single turnover” experiments, the ribozymes and DNA enzymes were used in a 10-fold excess. In “multiple turnover” experiments, the substrate mRNA was used in a 10-fold excess.
“Single Turnover” Kinetics
A kinetic analysis under “single turnover” conditions was carried out for the two most effective ribozymes and DNA enzymes (
“Multiple Turnover” Kinetics
Spinal nerve ligatures were placed as described by Kim & Chung (1992) on the left L5/L6 spinal nerves of 20 male Sprague-Dawley rats. At the same time, spinal catheters were implanted as described by Pogatzki et al. (2000). Four to six days after the operation, the tactile threshold baseline (withdrawal thresholds) was measured on the ipsilateral and contralateral hind paw using an electronic von Frey anaesthesiometer (IITC Life Science, USA). Correct positioning of the spinal catheter was confirmed by administering lidocaine (10 μl, 2%), which resulted in transient paralysis of both hind limbs. After the test and measurement of the baseline, 45 μg of VR-1 antisense oligonucleotides (AS, n=10) or mismatch oligonucleotides (MS, n=10) in 0.9% NaCl were in each case given once on the first day and b.i.d. on the following 4 days. The tactile withdrawal thresholds were measured 30 minutes after the first daily administration of oligo. The results are stated as a percentage of the maximum possible effect (% MPE) on the ipsilateral side, the base line being taken as 0% and the withdrawal threshold of a control group as 100% MPE. The antisense oligo used was V15 (
Treating mononeuropathic rats with antisense, but not with mismatch, oligodeoxynucleotides brought about a reduction in tactile allodynia beginning on the third day of treatment, with a plateau being reached on days 4 and 5 of treatment. There was no effect on the withdrawal thresholds of the contralateral hind paw.
Example 5 “Locked Nucleotides”/Oligonucleotides Protected from Degradation and Oligonucleotide ConstructsVarious different oligonucleotides according to the invention were produced, in particular according to subgroup (k) of
Various tests were carried out with these oligonucleotides:
a) Firstly, the percentage of RNA cleavage of VR1 by RNase H initiated by the oligonucleotide was investigated, the test conditions substantially matching those stated in Example 2.
The results are shown in Table 3. It was found that oligonucleotides with LNA only exhibited cleavage comparable with the native oligonucleotide if at least 6, or especially at least 8 contiguous nucleotides were not LNAs.
b) The melting temperature of the LNA/RNA:DNA hybrids were then measured by standard methods (Table 4). Surprisingly, the LNAs did not exhibit a raised melting temperature in comparison with the native oligonucleotides and the phosphorothioates. This is very favorable for stability.
c) The kinetics of RNase H cleavage were then investigated under identical conditions to those above, except that equimolar quantities of RNA and antisense oligonucleotide (100 nM each) were used. The results are shown in Table 5. Surprisingly, the oligonucleotide with LNA exhibited a distinct increase in activity in comparison with the native oligonucleotides and phosphorothioates.
d) Finally, the half-life of the radioactively labelled oligonucleotides with LNA of the native oligonucleotide and of the phosphorothioate was determined at 37° C. in human serum over a period of up to 2 days. The results are shown in Table 6. While the half-life of the native DNA is 1.5 h and that of the phosphorothioate is 10 h, the half-life of nucleotides with LNA, on the 3 or 4 ends, were significantly long with a t1/2 of approximately 17 h.
The best restriction properties were thus achieved by the nucleotides with LNAs which had approximately 8 contiguous nucleotides without LNAs and had 3 or 4 LNAs on the 3′ and 5′ ends.
The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.
BIBLIOGRAPHIC REFERENCES
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- Vaish, N. K. et al. (1998), Nucl. Acid Res. 26, 5237-5242.
Claims
1. An oligonucleotide comprising the sequence according to one of SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239.
2. An oligonucleotide according to claim 1, comprising
- (1) a base sequence according to SEQ ID NOs 28, 56, 73, 90, 107, 124, 141, 158, 169, 187, 204, 221, 232, or 249, or
- (2) a sequence differing from (1) by at most two different bases not located in the sequence according to SEQ ID NO 18, 46, 63, 80, 97, 114, 131, 148, 159, 177, 194, 211, 222, or 239, respectively.
3. An anti-sense oligonucleotide 15 to 30 nucleotides in length and comprising
- (1) the sequence according to one of SEQ ID NOs 19-27, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, or
- (2) a sequence differing from (1) by one different base not located in the sequence according to SEQ ID NO 18, 46, 63, 80, 97, 114, 131, 148, 159, 177, 194, 211, 222 or 239, respectively.
4. An anti-sense oligonucleotide according to claim 2, wherein the oligonucleotide is SEQ ID NO 28, 56, 73, 90, 107, 124, 141, 158, 169, 187, 204, 221, 232, or 249.
5. An anti-sense oligonucleotide selected from the group consisting of SEQ ID NOs 12-17, SEQ ID NOs 18-28, SEQ ID NOs 29-45, SEQ ID NOs 46-62, SEQ ID NOs 131-147, SEQ ID NOs 148-158, SEQ ID NOs 159-176, SEQ ID NOs 177-193, SEQ ID NOs 222-238, and SEQ ID NOs 239-249.
6. An oligonucleotide according to claim 5, wherein the oligonucleotide is selected from the group consisting of SEQ ID NOs 12-17, SEQ ID NOs 18-28, SEQ ID NOs 29-45, SEQ ID NOs 46-62, SEQ ID NOs 159-176 and SEQ ID NOs 177-193.
7. An oligonucleotide according to claim 5, wherein the oligonucleotide is selected from the group consisting of SEQ ID NOs 12-17, SEQ ID NOs 29-45 and SEQ ID NOs 159-176.
8. An oligonucleotide according to claim 5, wherein the oligonucleotide is selected from the group consisting of SEQ ID NOs 12-17 and SEQ ID NOs 29-45.
9. An oligonucleotide according to claim 1, wherein at least one of the nucleotides is a “locked nucleic acid” (LNA), and wherein more than 6 contiguous nucleotides in the oligonucleotide are not LNA's, and wherein the more than 6 contiguous nucleotides that are not LNAs are in the sequence domain of SEQ ID NO 18, 29, 46, 63, 80, 97, 114, 131, 148, 159, 177, 194, 211, 222 or 239.
10. A polynucleotide construct comprising at least one oligonucleotide according to claim 1.
11. A polynucleotide construct comprising, in two separate domains, Fragment I and Fragment III according to one of SEQ ID NOs 12-14, SEQ ID NOs 40-42, SEQ ID NOs 57-59, SEQ ID NOs 74-76, SEQ ID NOs 91-93, SEQ ID NOs 108-110, SEQ ID NOs 125-127, SEQ ID NOs 1452-144, SEQ ID NOs 170, 172 and 173, SEQ ID NOs 188-190, SEQ ID NOs 205-207, or SEQ ID NOs 233-235, or helix I and helix III according to one of SEQ ID NOs 15-17, SEQ ID NOs 43-45, SEQ ID NOs 60-62, SEQ ID NOs 77-79, SEQ ID NOs 94-96, SEQ ID NOs 11-113, SEQ ID NOs 128-130, SEQ ID NOs 145-147, SEQ ID NOs 174-176, SEQ ID NOs 191-193, SEQ ID NOs 208-210 or SEQ ID NOs 236-238.
12. A ribozyme comprising an oligonucleotide of claim 1, comprising helix I and helix III according to one of SEQ ID NOs 15-17, SEQ ID NOs 43-45, SEQ ID NOs 60-62, SEQ ID NOs 77-79, SEQ ID NOs 94-96, SEQ ID NOs 128-130, SEQ ID NOs 174-176 or SEQ ID NOs 191-193.
13. A ribozyme according to claim 12, comprising helix I and helix III according to SEQ ID NO 15, 43, 60, 77, 94, 128, 174 or 191.
14. A ribozyme according to claim 12, comprising helix I and helix III according to one of SEQ ID NOs 15-17, SEQ ID NOs 43-45 or SEQ ID NOs 174-176.
15. A ribozyme according to claim 12, comprising helix I and helix III according to one of SEQ ID NOs 60-62, SEQ ID NOs 94-96, SEQ ID NOs 128-130 or SEQ ID NOs 191-193.
16. A DNA enzyme comprising an oligonucleotide of claim 1, comprising Fragment I and Fragment III according to one of SEQ ID NOs 12-14, SEQ ID NOs 40-42, SEQ ID NOs 57-59, SEQ ID NOs 74-76, SEQ ID NOs 91-93, SEQ ID NOs 108-110, SEQ ID NOs 125-127, SEQ ID NOs 1452-144, SEQ ID NOs 170, 172 and 173, SEQ ID NOs 188-190 or SEQ ID NOs 205-207.
17. A DNA enzyme according to claim 16, comprising Fragment I and Fragment III according to one of SEQ ID NOs 12-14, SEQ ID NOs 40-42, SEQ ID NOs 108-110 or SEQ ID NOs 170, 172 and 173.
18. A DNA enzyme according to claim 16, comprising Fragment I and Fragment III according to one of SEQ ID NOs 57-59, SEQ ID NOs 91-93, SEQ ID NOs 125-127 or SEQ ID NOs 188-190.
19. A DNA enzyme according to claim 16, comprising Fragment I and Fragment III according to SEQ ID NO 14, 42, 59, 76, 93, 110, 127, 144, 173, 190 or 207.
20. A method for the treatment of pain, comprising administering an effective amount of a pharmaceutical composition comprising at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239, and a pharmaceutically acceptable carrier, to a patient in need thereof.
21. A method according to claim 20, for the treatment of chronic pain selected from the group consisting of tactile allodynia, thermally induced pain and inflammatory pain.
22. A method for the treatment of urinary incontinence, neurogenic bladder symptoms; pruritus; or tumors, comprising administering an effective amount of a pharmaceutical composition comprising at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239, and a pharmaceutically acceptable carrier, to a patient in need thereof.
23. A method for the treatment of vanilloid receptor subtype 1 (VR1)-associated disease, comprising administering an effective amount of a pharmaceutical composition comprising at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239, and a pharmaceutically acceptable carrier, to a patient in need thereof.
24. A method according to claim 23, wherein the VR1 receptor-associated disease is inflammation.
25. A gene therapy method comprising administering to a patient in need thereof an effective amount of an oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239.
26. A gene therapy method according to claim 25, which is an in vitro gene therapy.
27. A gene therapy method according to claim 25, which is an in vivo gene therapy.
28. A gene therapy method according to claim 25, wherein the oligonucleotide is comprised in an expression vector.
29. A gene therapy method according to claim 25, wherein the expression vector is comprised in a cell.
30. A process for the identification of pain-modulating substances comprising the following process steps:
- (a) providing a test cell which comprises at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239,
- (b) providing a control cell that does not comprise said oligonucleotide,
- (c) incubating a candidate substance under suitable conditions with the test cell, or a preparation made from the test cell, which has synthesized at least one vanilloid receptor,
- (d) measuring the binding of the test substance to the receptor synthesized by the test cell, or measuring at least one functional parameter of the receptor modified by binding of the test substance onto the receptor,
- (e) measuring the binding of the test substance to the receptor synthesized by the control cell, or measuring at least one functional parameter of the receptor modified by binding of the test substance onto the receptor,
- (d) identifying a substance by a difference between the measured value for the test cell and that for the control cell.
31. A method according to claim 30, wherein the vanilloid receptor is a VR-1 receptor.
Type: Application
Filed: Dec 23, 2009
Publication Date: Dec 16, 2010
Applicant: GRUENENTHAL GmbH (Aachen)
Inventors: Jens Kurreck (Berlin), Volker A. Erdmann (Berlin)
Application Number: 12/645,625
International Classification: A61K 48/00 (20060101); C07H 21/04 (20060101); C07H 21/02 (20060101); A61P 29/00 (20060101); A61P 35/00 (20060101); A61P 13/06 (20060101); A61P 17/04 (20060101); G01N 33/566 (20060101);