Treatment of pain.

(54) Title: TREATMENT OF PAIN. (54) Title: TREATMENT OF PAIN.

(57) Summary

Use of a therapeutic molecule, for the treatment of specific pain conditions, wherein the therapeutic molecule is a single chain polypeptide fusion protein, including: a non-cytotoxic protease, or a fragment thereof, such a protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a labeling portion that is capable of binding to a binding site on the nociceptive sensory afferent; such a binding site is capable of undergoing endocytosis that will be incorporated into an endosome within the nociceptive sensory afferent; a protease cleavage site in which the fusion protein is divisible by a protease, wherein the protease cleavage site is located between the protease or the nocytotoxic fragment thereof and the labeling portion; and a translocation domain that is capable of displacing the protease or protease fragment within an endosome, through the endosomal membrane, and into the cytosol of the nociceptive sensory afferent.

(57) Abstract

Use of a therapeutic molecule, for the treatment of specific pain conditions, where the therapeutic molecule is a single Chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorpofed into an endosome within fhe nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease, where the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; and a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent.

PATENT TITLE NO. 337729 _SE_

SSCRTORÍA IM ICÓMIMIA

Institute

Mexican Property

Industrial i

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Owner (s): SYNTAXIN LIMITED; ALLERGAN, INC.

Address: Units 4-10, The Quadrant, Barton Lañe, 0X14 3YS, Abingdon, Oxon, KINGDOM

UNITED

Denomination: PAIN TREATMENT.

Classification:

Inventor (s):

Int.CI.8: A61K38 / 48; A61K39 / 08; A61P25 / 04

KEITH FOSTER; JOHN CHADDOCK; PHILIP MARKS; PATRICK STANCOMBE; K. ROGER AOKI; JOSEPH FRANCIS; LANCE STEWARD

Number:

MX / y / 2008/0152% 7

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Country:

GB _t

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you

REQUEST

Int 'presentation date of June 2007

PRIORITY

Date:

June 2008

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Number:

0610867.4

Expiration Sheet

The reference patent is 1st with

In accordance with article 23 of the cc tada from the date; of presei dqpchos. yes tn subscribes the present title what h &lt;

Pi Industrial piety (Diario'Oficial de, __________

2M1 / 2004, 06/16/2005, 25 ^ 1/2006, 0 05 / 2009,06 / 01/20

a), 4th and 12th fractions I and

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with Federation (I

Insl Regulation of 2027) articles 1, 2 * section V. 6 * section III. and 59 of the Ustrial Property Law, this patent has a validity of twenty high taxes and I will be subject to the payment of the fee to keep industrial.

gables, the entities) 7/2002, 07/15/2004, 07/28/2004 and 39/2007); articles 1, 3,

Mexican Institute of industrial property (DOF 12/27/1999, refomiaoo 1 dt

OFBRÉSTOgBñSesTSüBcfirectores Dlvisfi Industrial. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

by articles 6 fractions III and 7^to bis 2 of Is .ey de la Stéformada on 08/02/1994, 10/25/1998, 12/26/1997, '/ 05/1999, ΐ / 08/2010 2 ^ / 01/2012 and 08/94 / 2012), articles 1^to, 3 * action V of IB Prooredad Industrial (DOF 14/12/1999, confirmed the V hateo eljBBIBonee I y III y 30 del Estatuí Orgánica

07/09/2004, 08/04/2004 and 09/13/2 17); one^to, 3

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Issue Date: March 16, 2016

THE DIVISIONAL-PATENT DIRECTOR

Í.'U. . ·

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NAHANNY CANAL REYES

Sand! No. 550, Floor 1,

Coi. Sania María Tepepan village. Xochsmiíco, C P. 16020,

Mexico City

Tea!. (55) 53 34 07 00 www.iinpi.goh.inx

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MX / 2016/22035

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PAIN TREATMENT

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Field of the Invention

The invention relates to the use of non-cytotoxic fusion proteins for the treatment of specific types of pain.

Background of the Invention

Toxins can generally be divided into two groups according to the type of effect they have on the target cell. In greater detail, the first group of toxins kill their natural target cells, and are therefore known as cytotoxic toxin molecules. This group of toxins is exemplified among other things by plant toxins such as castor bean and abrin, and bacterial toxins such as diphtheria toxin and exotoxins A from Pseudomonas. Cytotoxic toxins have attracted much interest in the design of "magic bullets" (eg immunoconjugates, which comprise a cytotoxic toxin and an antibody that binds to a specific marker in a target cell) for the treatment of cellular problems and conditions such as Cancer. Cictotoxic toxins typically kill their target cells by inhibiting the cellular process of protein synthesis.

The second group of toxins, which are known as non-cytotoxic toxins, (as the name implies) do not kill your natural target cells. Non-cytotoxic toxins have attracted much less commercial interest than their cytotoxic counterparts, and exert their effects on the target cell by inhibiting cellular processes other than protein synthesis. Non-cytotoxic toxins are

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produced by a variety of plants and by a variety of microorganisms such as Clostridium sp. and Neisseria sp.

Clostridial neurotoxins are proteins that typically have a molecular mass on the order of 150 kDa. They are produced by various species of bacteria, especially the Clostridium genus, most importantly C. tetani and various strains of C. botulinum, C. butyricum and C. argentinense. There are currently different classes of clostridial neurotoxins, in fact tetanus toxin and botulism neurotoxin in serotypes A, B, C1, D, E, F, and G, and they all share similar structures and modes of action.

Clostridial neurotoxins represent a major group of non-cytotoxic and sn toxin molecules synthesized by the host bacteria as single polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by means of a disulfide bond. The two chains are called the heavy chain (H chain), which has a molecular mass of approximately 100 kDa, and the light chain (L chain), which has a molecular mass of approximately 50 kDa.

L chains have a protease function (zinc-dependent endopeptidase activity) and have a high specificity for the substrate for the vesicle and / or plasma membrane-associated proteins involved in the exocytic process. The L chains of different clostridial species or

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INDUSTRIAL serotypes may have different hydrolase but specific peptide bonds in one of three substrate proteins, in fact slnaptobrevin, syntaxin, or SNPA-25. These substrates are important components of the neurosecretory machinery.

Neisseria sp., The most important of the N. gonorrhoeae species, produces functionally similar non-cytotoxic proteases. An example of such a protease is the IgA protease (see WO99 / ​​58571).

It has been well documented in the art that toxin molecules can be rerouted to a cell that is not the natural target cell for the toxin. When redirected in this way, the modified toxin is able to bind to the desired target cell, and after subsequent translocation into the cytosol, is able to exert its effect on the target cell. That redirection is accomplished by replacing the natural target (TM) portion of the toxin with a different TM. In this regard, the TM is selected such that it will bind to a desired target cell, and allow subsequent passage of the modified toxin into an endosome within the target cell. The modified toxin also comprises a translocation domain to allow entry of the non-cytotoxic protease into the cytosol of the cell. The translocation domain can be the natural translocation domain of the toxin or it can be a different translocation domain obtained from a microbial protein with translocation activity.

The aforementioned TM replacement can be carried out by means of conventional chemical conjugation techniques, which are

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The chemical conjugation, however, is frequently imprecise. For example, after conjugation a TM can bind to the rest of the conjugate at more than one point of attachment.

Chemical conjugation is also difficult to control. For example, a RTM can bind to the rest of the modified toxin at a point of attachment in the protease component and / or in the translocation component. This is problematic when binding to one of those components (preferably at a single site) is desired for therapeutic efficacy.

Thus chemical conjugation results in a mixed population of modified toxin molecules, which is undesirable.

As an alternative to chemical conjugation, TM replacement can be accomplished by recombinant preparation of a single polypeptide fusion protein (see EO98 / 07864). This Technique is based on the bacterial mechanism in vivo by means of which the native clostridia neurotoxin (this is holotoxin) is prepared, and results in a fusion protein that has the following configuration:

NH₂ - [protease component] - [translocation component] [TM] - COOH

According to WO98 / 07864, the TM is placed

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DE ΙΛ γ '.-' Ο.ΟΟλ ·.;. '(Οα.-TT? Towards the C-terminus of the TuS + ón-ta fusion protein is then activated by a protease drug, which cleaves at a site between the protease component and the translocation component. A two-chain protein is then produced, which comprises the protease component as a single polypeptide chain (via a disulfide bridge) to another single polypeptide chain containing the plus TM translocation component. While the methodology of WO98 / 07864 follows (in terms of the structural arrangement of the fusion protein) the clostridia holotoxin natural expression system, the present inventors have found that this system may result in the production of certain fusion proteins. which have substantially reduced binding ability for the target cell in question.

This problem is particularly relevant in the context of treating specific types of pain.

Brief Description of the Invention

The present invention addresses one or more of the aforementioned problems by providing the use of a therapeutic molecule for the manufacture of a medicament for the treatment of particular types of pain, wherein the therapeutic molecule is a chain polypeptide fusion protein a simple comprising:

a) a non-cytotoxic protease, or one or a fragment of it

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Likewise, such a protease or protease fragment is capable of dividing a protein from the TusToñ'éxócTfTca apparatus into a nociceptive sensory afferent;

b) a target portion that is capable of binding to a Binding Site in the nociceptive sensory afferent, such a Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent,

c) a protease cleavage site at the site at which the fusion protein is cleaved by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment and the target portion; and

d) a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, through the endosomal membrane and into the cytosol of the nociceptive sensory afferent.

The present inventors have found that the WO98 / 07864 fusion protein system is not optimal for TM requiring an N-terminal domain for interaction with a binding site in a nociceptive sensory afferent. This problem is particularly watered down with TMs that require a specific N-terminal amino acid residue or a specific sequence of amino acid residues that include the N-terminal amino acid residue

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In contrast to WO98 / 07864, the present invention employs non-cytotoxic fusion proteins, where the TM component of the fusion includes the relevant binding domain in an internal domain or in a mid-located amino acid sequence (this is the sequence linear peptide) of the TM, or preferably it is located towards the B terminal of the TM or more preferably in or near the N terminal. The B-terminal domain is capable of binding to the binding site in a nociceptive sensory afferent, and TM preferably has a requirement for a specific and defined sequence of amino acid residues in order for it to be free at its terminal

N.

The compounds described herein can be used to treat a patient suffering from one or more types of chronic pain including neuropathic pain, inflammatory pain, headache, somatic pain, visceral pain, and referred pain.

The term "treat" as used here means medically dealing with it. It includes, for example, administering a compound of the invention to prevent pain or reduce its severity.

The term "pain" as used here means an unpleasant sensory experience, usually associated with a physical disorder. The physical disorder may or may not be apparent to a doctor. Pain can be of two types: chronic and acute. A "sharp pain" is a short-term pain that has a sudden onset. A kind of

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Acute pain, for example, is skin pain felt when you hurt your skin and other superficial tissues, such as that caused by a cut or burn. Cutaneous nociceptors end just below the skin, and due to the high concentration of nerve terminals, produce well-defined localized pain of short duration. "Chronic pain" is pain other than acute pain. Chronic pain includes neuropathic pain, inflammatory pain, headache, somatic pain, visceral pain, and referred pain.

/. Neuropathic pain

The compounds of the invention can be used to treat pain caused by or otherwise associated with any of the following pain conditions. "Neuropathic pain" means abnormal sensory input, which results in discomfort of the peripheral nervous system, central nervous systems, or both.

A. Symptoms of neuropathic pain

Neuropathic pain symptoms may include spontaneous persistent pain, as well as allodynia (a painful response to a stimulus that is not normally painful), hyperalgesia (an accentuated response to a stimulus that usually causes only minor discomfort, such as a stinging needle), or hyperpathy (where a short discomfort turns into prolonged severe pain).

B. Causes of neuropathic pain

Neuropathic pain may be caused by any of the

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one. Traumatic insult, such as "eg efhp Io ~ uría ~ lési6η by compression of a nerve (eg, crushing a nerve, stretching a nerve, pinching a nerve, or incomplete transfection of a nerve); damage to the spinal cord (for example, hemisection of the spinal cord); amputation of a limb; a contusion; an inflammation (for example an inflammation of the spinal cord), or a surgical procedure.

2. An ischemic event including for example a stroke and heart attack.

3. An infectious agent.

Four. Exposure to a toxic agent, including for example a drug, an alcohol, a heavy metal (for example lead, arsenic, mercury), an industrial agent (for example a solvent, fumes from a glue) or nitrous oxide.

5. A disease including for example an inflammatory disease, a neoplastic tumor, acquired immunodeficiency syndrome (SSEQ ID A), Lyme disease or borreliosis, leprosy, a metabolic disease, a disease of peripheral nerves, such as a neuroma, a mini-neuropathy or a polyneuropathy .

C, Types of neuropathic disease

one. Neuralgia

Neuralgia is pain that radiates along the course of one or more specific nerves, usually without any demonstrable pathological changes in the nerve structure. The causes of

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Neuralgia are varied. Chemical irritation, inflammation, trauma (including surgery), understanding by nearby structures (for example tumors), and infections can all lead to neuralgia. In many cases, however, the cause is unknown or not identifiable. Neuralgia is more common in older people, but it can occur at any age. Neuralgia includes unlimited, thirtieth neuralgia, postherpetic neuralgia, postherpetic neuralgia, glossopharyngeal neuralgia, sciatica, and atypical facial pain.

Neuralgia is pain in the distribution of one or more nerves. Examples are thirtieth neuralgia, atypical facial pain, post-herpetic neuralgia (caused by shingles or herpes). The affected nerves are responsible for feeling touch, temperature, and pressure in the easy area from the jaw to the forehead. The problem can usually be described in a variety of ways such as "crippling", "sharp", "sudden", "burning", and even "burning" pain. In an atypical form of TN, pain can also be present as severe or just bothersome to last for long periods of time. The pain associated with TN is recognized as one of the most extreme pains that can be experienced.

Simple stimuli such as eating, talking, washing the face, or any light touch or feeling can trigger an attack (even the feeling of a gentle breeze). Attacks can occur in groups or as an isolated attack.

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Symptoms include acute paralyzing pain or constant burning pain located anywhere generally on or near the surface of the body, in the same location for each episode; pain along the path of the nerve; malfunction of the affected body part due to pain, or muscle weakness due to concomitant damage to the motor nerve; increased skin sensitivity or numbness of the affected skin area (sensation similar to a local anesthetic such as a novocaine injection); and any touch or pressure is interpreted as pain. Movement can also be painful.

Trigeminal neuralgia is the most common form of neuralgia. It affects the main sensory nerve of the face, the trigeminal nerve ("trigeminal" literally means "three origins", referring to the breakdown of the nerve into 3 branches). This condition includes sudden, short attacks of severe pain on one side of the face, along the area where the nerve is located. Attacks of pain can be severe enough to cause facial grimacing, which is classically referred to as a painful tic (tic douloureux). Sometimes the cause of trigeminal neuralgia, but a cause is not always identified. In this condition, certain movements such as chewing, talking, swallowing, or touching an area of ​​the face can trigger a spasm of extreme pain.

A related but rare neuralgia affects the glossal-pharyngeal nerve, which provides sensation to the throat. The

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Symptoms of this neuralgia are short, shock-like episodes of pain located in the throat.

Neuralgia can occur after infections such as shingles that is caused by the varicella-zoster virus, a type of herpes virus. This neuralgia produces constant burning pain after the itch has healed. Pain is worsened by movement or contact with the affected area. Not everyone diagnosed with shingles experiences postherpetic neuralgia, which can be more painful than shingles. Pain and tenderness can go on for months and even years. Pain usually takes the form of an intolerable sensitivity to touch but especially to light touch. Posttherpetic neuralgia is not restricted to the face; It can occur on any side of the body but usually occurs at the point where shingles is itchy.

Depression is common due to pain and social isolation during illness.

Post-herpetic neuralgia can be very long after the signs of the original herpes infection have disappeared. Other infectious diseases that can cause neuralgia are syphilis and Lyme disease.

Diabetes is another common cause of neuralgine. This very common medical problem affects almost 1 in 20 Americans during adulthood. Diabetes damages the small arteries that supply circulation to the nerves resulting in disease

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INDIA - ------ functioning of nerve fibers and sometimes loss of nerves. Diabetes can cause almost any neuralgia, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness of the hand and wrist), and paresthetic meralgia (numbness and pain in the thigh due to damage to the lateral femoral cutaneous nerve). Strict control of blood sugar can prevent nerve damage and can speed recovery in patients who develop neuralgia.

Other medical conditions that can be associated with neuralgia are chronic kidney failure and porphyria, an inherited disease in which the body cannot eliminate certain substances produced after the normal breakdown of blood in the body. Certain medications can also cause this problem.

2. Diaferentation

Diaferentation indicates a loss of sensory input to a portion of the body, and may be caused by disruption of peripheral sensory fibers or nerves of the central nervous system. A diaferentation pain syndrome includes without limitation damage to the brain or spine, post-embolism pain, phantom pain, paraplegia, branchial plexus avulsion damage, lumbar radiculopathies.

Four. Complex Regional Pain Syndromes (CRPS)

CRPS is the chronic pain syndrome resulting from sympathetically maintained pain and has two forms. CRPS 1

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CG L, \ - -,. T currently replaces the term "reflex sympathetic dystrophy syndrome". It is a chronic nervous problem that "" ~ occurs more frequently in the arms or legs after a minor or major injury. CRPS 1 is associated with severe pain; nail changes. Bones and skin; and a high sensitivity to touch of the affected limb. CRPS 2 replaces the term causalgia, and is the result of identified nerve damage. A CRPS includes without limit, a type I CRPS (reflex sympathetic dystrophy) and a type II CRPS (causalgia).

Four. Neuropathy

A neuropathy is a functional or pathological change in a nerve and is clinically characterized by sensory or motor neuron abnormalities.

Central neuropathy is a functional or pathological change in the central nervous system.

Peripheral neuropathy is a functional or pathological change in one or more of the peripheral nerves. Peripheral nerves carry information from your central nervous system (brain and spinal cord) to muscles and other organs, and from the skin, joints, and other organs back to the brain. Peripheral neuropathy occurs when those nerves do not carry information to and from the brain and spinal cord, resulting in pain, loss of sensation, or an inability to control muscles. In some cases, the inability of the nerves that control the blood vessels, intestines, and other organs result in pressure

INS'l'r. · 'Rro M ¿KiCANO D £ LA i' · '. ·: ·, IHJUüTUAL abnormal blood, digestion problems, and loss of other basic bodily processes. Risk factors for neuropathy include diabetes, high alcohol consumption, and exposure to certain chemicals and drugs. Some people have an inherited predisposition to neuropathy. Prolonged pressure on a nerve is another risk of developing a nerve injury. Pressure injury can be caused by prolonged immobility (such as a prolonged surgical procedure or prolonged illness) or compression of a nerve by casts, splints, braces, crutches, or other devices. Polyneuropathy involves an extensive process that usually affects both sides of the body equally. Symptoms depend on what type of nerve is affected. The three main types of nerves are sensory, motor, and autonomic. Neuropathy can affect one or a combination of all three types of nerves. Symptoms also depend on whether the combination affects the entire body or just one nerve (such as from injury). The cause of chronic inflammatory polyneuropathy is an abnormal immune response. Specific antigens, immune processes, and trigger factors are variable and in many cases are unknown. It can happen in association with other conditions such as HIV, irritable bowel disease, lupus erythematosus, chronic active hepatitis, and blood cell abnormalities. Peripheral neuropathy can include a pathological function or change of a single nerve or group of nerves

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Functional MPI or (mononeuropathy) or a multiple nerve change (polyneuropathy).

Peripheral neuropathies

Hereditary problems

Charcot-Marie-Tooth disease

Friedreich's ataxia

Systemic or metabolic diseases

Diabetes (diabetic neuropathy)

Diet deficiencies (especially vitamin B12) Excessive use of alcohol (alcoholic neuropathy) Uremia (due to kidney failure)

Cancer

Infectious or inflammatory conditions

SSEQ ID A

Hepatitis

Colorado Tick Fever

Diphtheria

Guillain Barre syndrome

HIV infection without development of SSEQ ID A Leprosy

Polyarteritis nodosa

Rheumatoid arthritis

Sarcoidosis

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Syphilis

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Systemic lupus erythematosus

Amyloid

Exposure to toxic compounds

Smelling glue or other toxic compounds

Nitrous oxide

Industrial agents - especially solvents

Heavy metals (lead, arsenic, mercury, etc.)

Neuropathy secondary to kidney disease by drugs such as pain relievers

Miscellaneous causes

Ischemia (decreased oxygenation / decreased blood flow)

Prolonged exposure to cold temperatures

to. Polyneuropathy

Polyneuropathy is a peripheral neuropathy that involves loss of movement or sensation to an area caused by damage or destruction of multiple peripheral nerves. Polyneuropathic pain includes, without limitation, post-polio syndrome, postmastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloid, toxins, SSEQ ID A, hypothyroidism, uremia, vitamin deficiencies, chemotherapy-induced pain, treatment with 2 ', 3'- dideoxycytidine (ddC), GuillainBarré syndrome or Fabry disease.

b. Mononeuropathy

Mononeuropathy is a peripheral neuropathy that includes loss of movement or sensation in an area caused by damage.

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or the destruction of a single peripheral nerve or group of nerves. Mononeurotherapy is most often caused by damage to a local area resulting from injury or trauma, although occasionally systemic problems can cause isolated nerve damage (as in the multiplex mononeuritis chaos). The usual causes are direct trauma, prolonged pressure on the nerve, and compression of the nerve due to inflammation or injury to neighboring body structures. Damage includes destruction of the myelin sheath (covering) of the nerve or part of the nerve cell (the axon). This damage slows or prevents impulse conduction through the nerve. Mononeuropathy can include any part of the body. Mononeuropathic pain includes, without limitation, a sciatic nerve dysfunction, a common peroneal nerve dysfunction, a radial nerve dysfunction, an ulnar nerve dysfunction, a VI cranial mononeuropathy, a Vil cranial mononeuropathy III (of the type of compression), cranial mononeuropathy III (diabetic type), auxiliary nerve dysfunction, carpal tunnel syndrome, femoral nerve dysfunction, tibial nerve dysfunction, Bell's palsy, thoracic narrow syndrome, carpal tunnel syndrome, and paralysis of the sixth (abducting) nerve.

c. Generalized peripheral neuropathies

Generalized peripheral neuropathies are symmetric, and are usually due to different systematic diseases and pathological processes that affect the peripheral nervous system in

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T Your entirety. They are also subdivided into various categories ---- i. Distal axonopathies are r es ü 11 a3o ^ Tré ™ ”aTgTrrras metabolic or toxic disturbances of neurons. They can be used for metabolic diseases such as diabetes, kidney failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. Distal axonoptía is a type of peripheral neuropathy that is the result of some metabolic or toxic discomfort of the neurons of the peripheral nervous system (PNS). It is the most common response of the nerves to metabolic or toxic disturbances, and as these can be caused by metabolic diseases such as diabetes, kidney failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy.

¡I. Myelinopathies are due to a primary attack on myelin causing acute conduction failure. The most common cause is acute inflammatory demyelinating polyneuropathy (ASEQ ID P; also known as Guillain-Barré syndrome), although other causes include chronic inflammatory demyelinating syndrome (CSEQ ID P), genetic metabolic problems (for example leukodystrophy), or toxins . Myelinopathy is due to the primary destruction of myelin or myelinating Schwann cells, leaving the axon intact but causing acute failure of impulse conduction. Demyelination reduces or blocks

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A completely nerve conduction. The most common inflammatory cause (PSIA, better known as Guillain Barré syndrome), although other causes include chronic inflammatory demyelinating polyneuropathy (CSEQ ID P), genetic metabolic problems (for example leukodystrophy or Charcot-Marie-Tooth disease), or toxins.

iii. Neuronopathies are the result of destruction of neurons of the peripheral nervous system (PNS). It can be caused by diseases of motor neurons, sensory neuropathies (eg herpes Zoster), toxins, or autonomic dysfunction. Neurotoxins can cause neuronopathies, such as vincristine from the chemotherapeutic agent. Neuropathy is a dysfunction due to damage to neurons of the peripheral nervous system (PNS), which results in peripheral neuropathy. It can be caused by motor neuronal diseases, sensory neuropathies (for example Herpes zoster), toxic substances or autonomic dysfunction. A person with nuronopathy can present it in different ways depending on the cause, the way it affects nerve cells, and the type of nerve cells that are most affected.

iv. Focal entrapment neuropathies (eg carpal tunnel syndrome).

II. Inflammatory pain

The compounds of the invention can be used to treat demyelinating Dolineurooatía

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MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY pain caused by or associated in any way with one of the following inflammatory conditions.

A. Arthritic problems

Arthritic problems include, for example, rheumatoid arthritis; juvenile rheumatoid arthritis; systemic lupus erythematosus (SLE); gouty arthritis; scleroderma; osteoarthritis; psoriasis arthritis; ankylosing spondylitis, Reiters syndrome (reactive arthritis), Still's disease in adults; arthritis from a viral infection; arthritis from a bacterial infection; such as for example gonococcal arthritis and non-gonococcal bacterial arthritis (septic arthritis); tertiary lyme disease; tuberculous arthritis, and arthritis from a fungal infection, such as blastomycosis.

B. Autoimmune diseases

Autoimmune diseases include for example Guillain-Barré syndrome, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyosistis, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome and Grave's disease.

C. Connective tissue disease

Connective tissue diseases include for example spondyloarthritis, dermatomyositis, and fibromyalgia.

D. Injuries

Inflammation caused by injury, including for example

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compression, perforation, stretching of a tissue or joint, can cause chronic inflammatory pain.

E. infection

Inflammation caused by infection including for example tuberculosis or interstitial keratitis can cause chronic inflammatory pain.

F. Neuritis

Neuritis is an inflammatory process that affects a nerve or group of nerves. Symptoms depend on the nerves in question, but can include pain, paresthesias, paresis, or hypersesthesia (numbness).

Examples include:

to. Branchial neuritis

b. Retrobulbar neuropathy, an inflammatory process that affects the part of the optic nerve that lies immediately behind the eyeball.

c. Optic neuropathy, an inflammatory process affecting the optic nerve that causes suddenly reduced vision in the affected eye. The cause of optic neuritis is unknown. Sudden inflammation of the optic nerve (the nerve that connects the eye and brain) leads to swelling and destruction of the myelin sheath. Inflammation can occasionally be the result of vial infection, or it can be caused by autoimmune diseases such as multiple sclerosis. Risk factors are related to possible causes.

<img file="MX337729B_D0025.tif" />

d. Vestibular neuritis, a viral infection that causes an inflammatory process that affects the vestibular nerve^-

G. Inflammation of the joints

Inflammation of the joints such as those caused by bursitis or tendonitis, for example, can cause chronic inflammatory pain.

III. Headache

The compounds of the invention can be used to treat pain caused by or otherwise associated with any of the following headache conditions. A headache (medically known as celphalgia) is a condition of moderate to severe pain in the head; sometimes neck and upper back pain can be interpreted as headache. It can indicate a local or systemic disease or be a disease by itself.

A. Muscle / myogenic headache

Muscle / myogenic headaches involve tightening or tension of the neck and acial muscles; they can radiate to the forehead. Tension headache is the most common form of myogenic headache.

A tension headache is a condition that involves pain or discomfort in the head, neck, or neck, usually associated with muscle stiffness in those areas. Tension headaches can be the result of contraction of the neck and neck muscles. One cause of this muscle contraction is a response to

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tension, depression or anxiety. Any activity that causes the head to stay in one position for a long period without moving can cause a headache. Those activities include typing or using computers, fine handwork, and using a microscope. Sleeping in a cold room or sleeping with your neck in an abnormal position can also cause this type of headache. A tension-type headache includes, without limitation, an episodic tension headache and a chronic tension headache.

B. Vascular headache

The most common type of vascular headache is migraine. Other types of vascular headaches include cluster headaches, which cause repeated episodes of severe pain, and headaches that result in high blood pressure.

one. Migraine

A migraine is a heterogeneous problem that generally includes recurrent headaches. Migraines are different from other headaches because they occur with other symptoms, such as nausea, vomiting, or sensitivity to light. In most people, throbbing pain is felt only on one side of the head. Clinical features such as the type of aura symptoms, the presence of prodomos, or associated symptoms such as vertigo can be observed in groups of patients with different pathophysiological and genetic mechanisms. A migraine pain include unlimited, a migraine without aura (common migraine), a

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2. Cluster headache

Cluster headache affects one side of the head (unilateral) and can be associated with watery eyes and nasal congestion. It occurs in clusters, passing repeatedly each day at the same time for several weeks and then remitting.

D. Hypertension headache

E. Traction headache and inflammation

Traction and inflammation headaches are usually symptoms of other problems, ranging from ischemia to sinusitis.

F. Hormonal headache

G. Rebound headache

Rebound headaches, also known as overuse headaches, occur when the medicine is taken too frequently to relieve the headache. Rebound headaches frequently occur daily and can be very painful.

H. Headache from chronic sinusitis

Sinusitis is a bacterial, fungal, viral, allergic or autoimmune inflammation of the paranasal sinuses. Chronic sinusitis is one of the most common complications of the common cold. Symptoms include nasal congestion; easy pain; headache

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fever; general discomfort; thick yellow or green discharge feeling of facial fullness that worsens on flexing. In a small number of cases, chronic maxillary sinusitis can also present as bacteria from a dental infection spread. Chronic hyperplastic eosinophilic sinusitis is a noninfectious form of chronic sinusitis.

I. Organic headache

J. Ictal headaches

Ice headaches are headaches associated with seizure activity.

IV. Somatic pain

The compounds of the invention can be used to treat pain caused by or otherwise associated with any of the following somatic pain conditions. Somatic pain originates from ligaments, tendons, bones, blood vessels, and even the nerves themselves. It is detected with somatic nociceptors. The paucity of pain receptors in these areas produces mild, less localized pain that lasts longer than cutaneous pain; Examples include dislocations and bone fractures. Additional examples include the following:

A. Excessive muscle tension

Excessive muscle tension can be caused, for example, by spraining or pulling.

B. Repetitive motion problems

Repetitive motion problems can be the

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C. Muscle problems

Muscle problems causing somatic pain include for example polymyositis, dermatomyositis, lupus, fibromyalgia, polymyalgia rheumatica, and rhabdomyolysis.

D. Mialgia

Myalgia is muscle pain and is a symptom of many diseases and problems. The most common cause of myalgia is either overuse and overstretching of a muscle or group of muscles. Myalgia without a traumatic history is frequently due to viral infections. Prolonged myalgias may indicate metabolic myopathy, some nutritional deficiencies, or chronic fatigue syndrome.

E. infection

The infection can cause somatic pain. Examples of such infections include, for example, muscle abscesses, trichinosis, influenza, Lyme disease, malaria, rocky mountain fever, avian influenza, the common cold, community-acquired pneumonia, meningitis, smallpox, acute respiratory syndrome severe, toxic shock syndrome, typhoid fever, and upper respiratory tract infection.

F. Drugs

Drugs can cause somatic pain. Those drugs

<img file="MX337729B_D0029.tif" />

They include for example cocaine, a statin to lower cholesterol (such as atorvastatin, simvastatin, and lovastatin), and a CE inhibitor to lower blood pressure (such as enalapril and captopril).

V. Visceral pain

The compounds of the invention can be used to treat pain caused by or associated with any of the visceral pain conditions. Visceral pain originates from the viscera or organs of the body. Visceral nociceptors are located within the internal organs and cavities. The even greater shortage of nociceptors in these areas produces pain that is usually more acute and of longer duration than somatic pain. Visceral pain is extremely difficult to locate, and various damage to visceral tissue presents “referred” pain, where the sensation is located in an area completely unrelated to the site of injury. Examples of visceral pain include the following.

A. Functional visceral pain

Functional visceral pain, for example irritable bowel syndrome and chronic functional abdominal pain (CFAP), functional constipation and functional dyspepsia, noncardiac chest pain (CNP), and chronic abdominal pain.

B. Chronic gastrointestinal inflammation

Chronic gastrointestinal inflammation include for example gastritits, inflammatory bowel disease such as Crohn's disease, ulcerative colitis, microscopic colitis,

<img file="MX337729B_D0030.tif" />

diverticulitis and gastroenteritis; interstitial cystitis, intestinal ischemia; cholecystitis, appendicitis, gastroesophageal reflux, ulcer, nephrolithiasis, urinary tract infection, pancreatitis and hernia.

C. Autoimmune pain

Autoimmune pain includes for example sarcoidosis and vasculitis.

D. Organic visceral pain

Organic visceral pain includes, for example, pain resulting from traumatic, inflammatory, or degenerative injury to the viscera, or caused by a tumor impacting sensory innervation.

E. Treatment-induced visceral pain

Treatment-induced visceral pain includes, for example, pain due to chemotherapy or radiation therapy.

SAW. Referred pain

The compounds of the invention can be used to treat pain caused by or associated in any other way with any of the following reported pain conditions.

Referred pain arises from pain located in a separate area from the site of the pain stimulus. Often referred pain occurs when a nerve is compressed or damaged at or near its origin. In this circumstance, the sensation of pain will generally be felt in the territory that the nerve serves, although the damage originates elsewhere. A common example occurs in the herniated

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intervertebral disc, in which a nerve root emerging from the spinal cord is compressed by adjacent disc material. Although pain may arise in the damaged disc itself, the pain will also be felt in the region served by the compressed nerve (eg, thigh, knee, or foot). Relief of pressure on the nerve root may improve the referred pain, provided that permanent nerve damage has not occurred. Myocardial ischemia (loss of blood flow to part of the heart muscle tissue) is possibly the best-known example of the referred pain; the sensation may occur in the upper chest as a restricted sensation, or as pain in the shoulder, arm, or even the left hand.

The non-cytotoxic component of the protease of the present invention is a non-cytotoxic protease, or a fragment thereof, protease or the protease fragment that is capable of cleaving different but specific peptide bonds into one of three substrate proteins , namely synaptobrevin, syntaxin or SNAP25, of the exocytic apparatus of fusion in a nociceptive sensory afferent. These substrates are important components of the neurosecretory machinery. The non-cytotoxic component of the protease of the present invention is preferably a neisseria IgA protease or a fragment thereof or a Clostridia neurotoxin L chain or a fragment thereof. A not particularly preferred cytotoxic component of protease is a botulinum neurotoxin (BoNT) L chain or a fragment

<img file="MX337729B_D0032.tif" />

Of the same. '

The translocation component of Ta pTesFirtT ^ mveTrcTórT allows translocation of the non-cytotoxic protease (or its fragment) into the target cell such that functional expression of protease activity occurs within the cytosol of the target cell. The translocation component is preferably capable of forming ion-permeable pores in lipid membranes under low pH conditions. It has preferably been found that only those portions of the protein molecule capable of forming pores within the endosomal membrane have been used. The translocation component can be obtained from a microbial protein source, particularly from a bacterial or viral protein source. Therefore, in one embodiment, the translocation component is a displacement domain of an enzyme, such as a bacterial toxin or a viral protein. The translocation component of the present invention is preferably an clostridia neurotoxin H chain or a fragment thereof. More preferably it is the HN domain (or a functional component thereof), where HN means a portion or fragment of the H chain of a clostridial neurotoxin approximately equivalent to half of the amino terminus of the H chain, or the domain which corresponds to that fragment in the intact H chain.

The TM component of the present invention is responsible for binding the fusion protein of the present invention to a point

<img file="MX337729B_D0033.tif" />

link in a target cell. Thus, the TM component is simply a ligand through which a fusion protein of the present invention binds to a selected target cell.

In the context of the present invention, the target cell is a nociceptive sensory afferent, preferably a primary nociceptive afferent (eg, an A fiber such as a Αδ fiber or a C fiber). Thus, the fusion proteins of the present invention are capable of inhibiting neurotransmitter or neuromodulator release [eg glutamate, substance P, calcitonin gene related peptide (CGRP), and / or neuropeptide Y] from discrete populations of nociceptive sensory afferent neurons. In practice, fusion proteins reduce or prevent the transmission of sensory afferent signals (for example neurotransmitters or neuromodulators) from peripheral or central pain fibers, and therefore can be applied as therapeutic molecules for the treatment of pain, particularly chronic pain.

It is routine to confirm that TM binds to a nociceptive sensory afferent. For example, a simple radioactive displacement experiment can be employed in which the tissue or cell representative of the nociceptive sensory afferent (eg DRG) is exposed to the labeled ligand (eg tritiated) in the presence of excess ligand without label. In such an experiment, the relative proportions of non-specific and specific binding can be determined, thereby allowing the

<img file="MX337729B_D0034.tif" />

confirmation that the ligand binds to the nociceptive sensory afferent target cell. Optionally, the analysis may include one or more binding antagonists, and the analysis may further encompass observation of a loss of ligand binding. Examples of this type of experiment can be found in Hulme, EC (1990), Receptor Binding Studies, A Brief Review, pgs. 303-311, in In Receptor biochemistry, A Practical Approach, Ed. EC Hulme, Oxford University Press.

The fusion proteins of the present invention generally show a reduced binding affinity (in the range of up to one hundred fold) for nociceptive sensory afferent target cells compared to corresponding free TM. However, despite this observation, the fusion proteins of the present invention show surprisingly good efficacy. This can be attributed to two main characteristics. First, the non-cytotoxic component of protease is catalytic - thus, the therapeutic effect of some such molecules is rapidly amplified. Second, the receptors present in nociceptive sensory afferents need only act as a pathway for therapeutic entry, and need not necessarily be stimulated to a level required to achieve a ligand receptor-mediated pharmacological response. Accordingly, the fusion proteins of the present invention can be administered in a dosage that is much lower than that which would be employed for other types of analgesic molecules.

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such as NSASEQ ID S, morphine, gabapentin. These latter molecules are typically administered in amounts of several micrograms to milligrams (even up to hundreds of milligrams), while the fusion proteins of the present invention can be administered in much lower dosages, typically at least ten times less, and more typically a hundred times less.

TM preferably includes a maximum of 50 amino acid residues, preferably a maximum of 40 amino acid residues, particularly preferably a maximum of 30 amino acid residues, and more preferably a maximum of 20 amino acid residues.

Opiates represent a preferred group of TMs of the present invention. Within this family of peptides are included enkephalins (met and leu), endomorphins 1 and 2, βendorphins and dynorphin. Opioid peptides are frequently used clinically to modify activity to nociceptors, other cells involved in the pain response. As exemplified by the World Health Organization's three-stage analgesic scale, opiates have entry points into the pharmacological treatment of cancer pain and not cancer in all three stages, underscoring its importance in pain management. . The reference to opiates encompasses their fragments, derivatives, variants, which retain the ability to bind to nociceptive sensory afferents.

<img file="MX337729B_D0036.tif" />

The TM of the invention can also be a molecule that acts as an agonist in one or more of the receptors present in a nociceptive sensory afferent, more particularly in a primary nociceptive afferent. Conventionally, an agonist has been considered any molecule that can increase or decrease activities within a cell, namely any molecule that simply causes an alteration in cell activity. For example, the conventional meaning of an agonist would include a chemical capable of combining with a receptor in a cell and initiating a reaction or activity, or a drug that induces an active response by activating the receptors, if the response is an increase or a decrease in cellular activity.

However, for the purposes of this invention, an agonist is more specifically defined as a molecule that is capable of stimulating the process of exocytic fusion in a target cell, that the process is susceptible to inhibition by a protease (or a fragment thereof) capable of dividing a protein from the exocytic fusion apparatus in the target cell.

Accordingly, the particular definition of the agonist of the present invention would exclude many molecules that would be conventionally considered to be agonists. For example, nerve growth factor (NGF) is an agonist in terms of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when determined by the above criteria because it does not

<img file="MX337729B_D0037.tif" />

it is a major inducer of exocytic fusion. Furthermore, the process that NGF stimulates (ie cell differentiation) is not susceptible to inhibition by protease activity of a non-cytotoxic toxin molecule.

The characteristics of the TM agonist that binds to a receptor on a nociceptive afferent can be confirmed using the methods described in Example 10.

In a preferred embodiment of the invention, the target for TM is the ORLi receptor. This receptor is a member of the class of G-protein coupled receptors, and has a seven transmembrane domain structure. The properties of the ENT receptor₁ they are discussed in detail by Mogil &amp; Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages 381-415.

In one embodiment, TM is a molecule that binds (preferably specifically binds) to the ENT receptor_VPreferably TM is an ORL · receptor agonist ,. The term "agonist in this context is defined as above.

The characteristics of the TM agonist that binds to an ORLt receptor can be confirmed using the methods described in Example 10. These methods are based on previous experiments [see Inoue et al. 1998 [Proc. National. Acad. Sci. 95, 10949-10953]), confirming that the natural ORL-ι receptor agonist, nociceptin, causes the induction of substance P release from nociceptive primary afferent neurons. This is supported by the fact that:

<img file="MX337729B_D0038.tif" />

&gt; nociceptin-induced responses are suppressed by specific receptor antagonists ^ NKI (the substance P receptor); and&gt; pretreatment of cells with capsicin (which depletes substance P from small-diameter primary afferent neurons) attenuates nociceptin-induced responses.

Similarly, Inoue et al. confirm that an intraplantar injection of botulinum type A neurotoxin suppresses nociceptin-induced responses. Since BoNT is known to

Inhibits the release of substance P from primary afferent neurons (Welsh et al, 2000, Toxicon, 38, 245-258), this confirms the coupling between the subsequent release of the interaction and the nociceptin-ORLí of substance P

Thus, a TM may be said to have agonist activity at the ORLj receptor if TM causes an induction in the release of substance P from a sensory afferent neuron noclceptlva (see Example 10).

In a particularly preferred embodiment of the invention, TM is nociceptin - the natural ligand for the ORLi receptor. Nociceptin targets the ENT receptor! with high affinity. Examples of the other preferred TMs include:

<td>Code</td><td>Sequence</td><td>Ref.</td><td>SEQ IO No.</td>

<td>Noniceptin 1-17</td><td>FGGFTGARKSARKLANQ</td><td> [1]</td><td> 37,38</td>

<td>Noniceptin 1-11</td><td>FGGFTGARKSA</td><td> [1]</td><td> 39,40</td>

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<td>Noniceptin Y10jl-11</td><td>FGGFTGARKYA</td><td>m</td><td> 41,42</td>

<td>Noniceptin [Y11] 1-11</td><td>FGGFTGARKSY</td><td>gave</td><td> 43,44</td>

<td>Ñoniceptin [Y14J1-17</td><td>FGGFTGARKSARKYANG</td><td>(1J</td><td> 45,46</td>

<td>Noniceptin 1-13</td><td>FGGFTGARKSARK</td><td> (2)</td><td> 47,48</td>

<td>Noniceptin [R14K15] 1- 17 (also known in this description as variant noniceptin</td><td>FGGFTGARKSARKRKNQ</td><td> [3,4]</td><td> 49,50</td>

<td>Peptide agonist</td><td>Peptide agonists of the library method combination</td><td>[YES</td><td></td>

[1] Mogil &amp; Pasternak, 2001, Pharmacol. Rev., 53, 381-415 [2] Maile et al., 2003, Neurosci. Lett., 350, 190-192 [3] Rizzi et al., 2002, J. Pharmacol. Exp. Therap., 300, 57-63 [4] Okada et al., 2000, Biochem. Biophys. Res. Commun., 278, 493498 [5] Dooley et al., 1997, J Pharmacol Exp Ther. 283 (2), 735-41.

The previously identified variant of TM shows particularly good binding affinity (compared to natural nociceptin) for nociceptive sensory afferents. This is amazing as amino acid modifications occur in one position.

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In the context of the present invention, the term opioid or ENT receptor agonist! (such as nociceptin, or any of the peptides listed in the table above) that have at least 70%, preferably at least 80%, preferably at least 90%, and more preferably at least 95% homology with the opiate or agonist. Agonist homologs retain the characteristics of the nociceptin agonist at the ORL! Receptor, which can be tested using the methods

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provided in Example 10. Similarly, an opioid homolog substantially retains the binding function of the opioid with which it exhibits high homology.

The invention also encompasses the fragments, variants, derivatives of any TM described above. These fragments, variants, derivatives substantially retain the characteristics attributed to TM.

In addition to the aforementioned non-opioid or opioid classes of TM, a variety of other polypeptides are suitable for targeting the fusion proteins of the present invention to nociceptive sensory afferents (eg, nociceptors). In this regard, particular reference is made to galanin and galanin derivatives. Galanin receptors are found post- and pre-synaptic in DRG (Liu &amp; Hokfelt, (2002), Trends. Pharm. Sci., 23 (10), 468-74), and expression is improved during states of neuropathic pain. Proteinase activated receptors (PAR) are also a preferred group of TMs of the present invention, in particular PAR-2. PAR-2 ​​agonists are known to induce / elicit acute inflammation, in part via a neurogenic mechanism. PAR2 is expressed by the primary spinal afferent neurons, PAR2 agonists stimulate the release of substance P (SP) and of the calcitonin gene related peptide and (CGRP) in peripheral tissues.

A particularly preferred TM system of the present invention includes:

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<td>Flirting</td><td>Reference</td>

<td>Noniceptin</td><td>Guerrini, ota /., (1997) J Med, Chem., 40, pp. 178 ^ f7S3</td>

<td>β-endorphin</td><td>Btene, et al., (1983) J. Biol. Chem., 258 (13), pp. 8277-8284</td>

<td>Endomorphine-1;</td><td>Zaina »of ai, (1997). Nature, 388, pp.</td>

<td>Endomorphine-2</td><td> 499-502</td>

<td>Dynorphine</td><td>Fiélds &amp; Baabaum (2002) chapter 11, In The Textbook of Rain, Wall &amp; Melzack eds.</td>

<td>Met-enkephalin</td><td>Laugh &amp; Baabaum (2002) chapter 11, In The Textbook of Pain, Wall &amp; Melzack eds.</td>

<td>Leu-enkephalin</td><td>Fields &amp; Baabaum (2002) chapter 11, In The Textbook of Paín, Wall &amp; Melzack eds.</td>

<td>Galanina</td><td>Xu ef ai, (2000) Neuropaptides, 34 (384), 137-147</td>

<td>PAR-2 ​​peptide</td><td>Vergnoíle et al., (2001) Nat. Med., 7 (7), 821-826</td>

The protease cleavage site of the present invention allows cleavage (preferably controlled cleavage) of the fusion protein at a position between the non-cytotoxic protease component and the TM component. It is this cleavage reaction that converts the fusion protein from a single-chain polypeptide to a disulfide-linked double-chain polypeptide.

According to a preferred embodiment of the present invention, TM

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it is linked via a domain or amino acid sequence that is located far from the C-terminus of TM. For example, the relevant liganTé ”domain may include an inner domain or an amino acid sequence midway (ie the linear peptide sequence) of TM. Preferably, the relevant link domain is located towards the N terminal of TM, preferably at or near terminal N.

In one embodiment, the single-chain polypeptide fusion can include more than one proteolytic cleavage site. However, when two or more such sites exist, they are different, thus preventing the occurrence of multiple disruption events in the presence of a single protease. In another embodiment, it is preferred that the polypeptide single chain fusion have a single protease cleavage site.

Protease cleavage sequences can be introduced (and / or any inherent cleavage sequences can be removed) at the DNA level by conventional means, for example by site-directed mutagenesis. The investigation to confirm the presence of break sequences can be carried out manually or with the help of computer programs (for example the MapDraw program by DNASTAR, Inc.).

While any site of protease cleavage can be employed, the following are preferred:

Enterokinase

Factor Xa (DDDDKJ.) (IEGR | / SEQ ID GRj)

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TEV (tobacco mosaic virus) (ENLYFQjG)

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PreScission (LEVLFQj, GP).

Also included in the term protease cleavage site is an intein, which is an auto-cleavage sequence. The self-splicing reaction is controllable, for example by varying the concentration of the reducing agent present.

During operation, the protease cleavage site is ruptured and the N-terminal region (preferably the N-terminal) of TM is exposed. The resulting polypeptide has a TM with an N-terminal domain or an internal domain that is substantially free of the residue of the fusion protein. This arrangement ensures that the N-terminal component (or inner domain) of the TM can interact directly with a binding site in a target cell.

In a preferred embodiment, the TM and the protease cleavage site are spaced from the fusion protein by at most 10 amino acid residues, preferably at most 5 amino acid residues, and most preferably by zero amino acid residues. Thus, after cleavage of the protease cleavage site, a fusion is provided with a TM having an N-terminal domain that is substantially free of the remainder of the fusion.

This arrangement ensures that the half-range N-terminal component can reciprocate directly with a link point in a target cell.

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An advantage associated with the activation step previously stated is that TM only becomes susceptible to degradation of the N-terminus once the proteolytic cleavage of the fusion protein has occurred. Furthermore, selection of a specific protease cleavage site allows selective activation of the fusion of the polypeptide in a double-stranded conformation.

The single-chain polypeptide fusion construct of the present invention places the site of the protease cleavage between TM and the non-cytotoxic component of protease.

It is preferred that, in the single chain fusion, TM is located between the protease cleavage site and the translocation component. This ensures that TM is linked to the translocation domain (i.e. as occurs with native clostridial holotoxin), although in the case of the present invention the order of the two components is reversed versus native holotoxin. Another advantage with this arrangement is that TM is located in an exposed region of the fusion protein loop, which has minimal structural effects on the conformation of the fusion protein. In this regard, carcass is variously referred to as the linker, the activation link, the inter-domain linker, or just the surface exposed link (Schiavo et al 2000, Phys. Rev., 80, 717-766; Turton et al. , 2002, Trends Biochem. Sci., 27, 552-558).

In one embodiment, in the single chain polypeptide, the non-cytotoxic protease component and the translocation component are linked via a disulfide bond. So,

I Μ. ΡI after cleavage of the protease cleavage site, the polypeptide assumes a double-stranded conformation, where the protease translocation components are still linked together by the disulfide bond. For this purpose, it is preferred that the translocation components of the protease are spaced apart from each other in the single chain fusion protein by a maximum of 100 amino acid residues, preferably a maximum of 80 amino acid residues, particularly preferred by a maximum of 60 amino acid residues, and more preferably by a maximum of amino acid residues.

In one embodiment, the non-cytotoxic component of protease forms a disulfide bond with the translocation component of the fusion protein. For example, the amino acid residue of the protease component that forms the disulfide bond is located within the last 20, preferably within the last 10 amino acid residues of the C-terminal of the protease component. Similarly, the amino acid residue within the translocation component that forms the second part of the disulfide bond can be located within the first 20, preferably within the first 10 amino acid residues of the N-terminus of the translocation component.

Alternatively, in the single-chain polypeptide, the non-clototoxic component of protease and the TM can be linked together by a disulfide bond. In this regard, the TM amino acid residue that forms the disulfide bond is preferably located far

<img file="MX337729B_D0049.tif" />

from terminal N of TM, preferably towards terminal C of

TM.

In one embodiment, the non-cytotoxic component of protease forms a disulfide bond with the TM component of the fusion protein. In this regard, the amino acid residue of the protease component that forms the disulfide bond is preferably located within the last 20, preferably within the last 10 residues of the C-terminal amino acid of the protease component. Similarly, the amino acid residue within the TM component that forms the second part of the disulfide bond is preferably located within the last 20, preferably within the last 10 C-terminal amino acid residues of TM.

The above disulfide bond arrangements have the advantage that the translocation components of the protease are arranged similarly to the native clostridial neurotoxin. By way of comparison, referring to the primary amino acid sequence for the native clostridial neurotoxin, the respective cysteine ​​amino acid residues are spaced from each other between 8 and 27 amino acid residues - taken from Popoff, SR. &amp; Marvaud, JC, 1999, Structural &amp; genomic features of clostridial neurotoxins, chapter 9, in The Comprehensive Sourcebook of Bacterial Protein

Toxins. Ed. Alouf &amp; Freer:

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<td>Serotype ¹</td><td>Sequence</td><td>Native length between CC</td>

<td>80NT / AI</td><td>CVRGIITSKTKS -— LDKGYNKALNDLC</td><td> 23</td>

<td>BQNT / A2</td><td>CVRGIIPFKTKS — LDEGYNKALNDLC</td><td> 23</td>

<td>BoNT / B</td><td>CKSVKAPG -........ IC</td><td> 8</td>

<td>BoÑT / C</td><td>CHKAIDGRS —— LYNKTLDC</td><td> 15</td>

<td>BoÑT / D</td><td>CLRLTK —-—- ÑSRDDSTC</td><td> 12</td>

<td>BoÑT / E</td><td>'CKÑ-IVSVK —-— GIRK — SlC</td><td> 13</td>

<td>BoNT / F</td><td>CKS-VIPRK --------- GTKAPP-RLC</td><td> 15</td>

<td>BoÑTTG</td><td>CKPVMYKÑT ------- GKSE — GC</td><td> 13</td>

<td>Tent</td><td>CKKIÍPPTNIRENLYNRTASLTDLGGÉLC</td><td> 27</td>

information only on proteolytic strains

The fusion protein may include one or more purification tags, the N-terminus of which is located on the protease component and / or the C-terminal on the translocation component.

While any purification label can be used, the following are preferred:

His tag (eg 6 x histidine), preferably as the C-terminal and / or the N-terminal tag of MBP (maltose binding protein), preferably as the N-terminal tag of GST (glutathione-S-transferase), preferably as the Terminal N label His-MBP label preferably as Terminal N label,

<img file="MX337729B_D0050.tif" />

GST-MBP tag preferably as Terminal N tag, thioredoxin tag preferably as Terminal N tag, CBD (chitin binding domain) tag preferably as Terminal N tag.

In accordance with another embodiment of the present invention, one or more molecules of the peptide spacer can be included in the fusion protein. For example, a peptide spacer can be used between a purification tag and the rest of the fusion protein molecule (for example between a N-terminal purification tag and a component of the protease of the present invention; and / or between a purification C · terminal tag and a translocation component of the present invention). A peptide spacer can also be used between TM and the translocation components of the present invention.

A variety of various spacer molecules can be employed in any of the fusion proteins of the present invention. Examples of such spacer molecules include those illustrated in Figures 28 and 29. Particular mention is made here of GS15, GS20, GS25, and Hx27 - see Figures 28 and 29.

The present inventors have unexpectedly found that the fusion proteins (eg. CPNv / A) of the present invention can show improved binding activity for nociceptive sensory afferents when the size of the spacer is selected such that (in use) the terminal C of TM and the χΛ Ρ ϊ

INSTITUTE V; -. 0 ·? Ι ds la ?? &lt;&gt; .-; .-.?. · 3 \

Terminal N of the translocation component was separated W érttre '^ rpó /

Tac (hybrid)

AraBAD

40-105 angstroms, preferably by 5 0 -1U 0 angsT7OTTfS7''ynTáS 'preferably by 50-90 angstroms. In another embodiment, the preferred spacers have an amino acid sequence of 11-29 amino acid residues, preferably 15-27 amino acid residues, and more preferably 20-27 amino acid residues. Convenient spacers can be routinely identified and obtained according to Crasto, CJ. and Feng, JA (2000) May 13 (5), pgs. 309-312 - see also http: //www.fccc/edu/research/labs/feng/limker.html.

In accordance with a second aspect of the present invention, a DNA sequence encoding the aforementioned single chain polypeptide is provided. In a preferred aspect of the present invention, the DNA sequence is prepared as part of a DNA vector, wherein the vector encompasses a promoter and a terminator.

In a preferred embodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition

IPTG 0.2mm (0.05-2.0mM)

L-arabinose 0.2% (0.002-0.4%) operator T7-lac IPTG 0.2 mM (0.05-2.0mM)

The DNA construct of the present invention is preferably designed in silico, and then synthesized by conventional DNA synthesis techniques.

<img file="MX337729B_D0051.tif" />

The above DNA sequence information is optionally modified for codon predisposition according to the most modern host cell expression system (eg Escherichia Coli) to be employed.

The DNA base is preferentially analyzed for any sequence inherent in nucleic acid, which when transcribed and translated would produce an amino acid sequence corresponding to the protease cleavage site encoded by the second peptide coding sequence. This research can be done manually or with the help of computer programs (for example the MapDraw program by ADNSTAR, Inc.).

In accordance with another embodiment of the present invention, there is provided a method for preparing a non-cytotoxic agent, including:

to. contacting a single chain polypeptide fusion protein of the invention with a protease capable of disrupting the protease cleavage site:

b. breaking the protease break site, and so forming a double chain fusion protein.

This aspect provides a double chain polypeptide, which generally mimics the structure of clostridial holotoxin. In more detail, the resulting double chain polypeptide typically has a structure where:

to. the first chain encompasses the non-cytotoxic protease, or a fragment thereof, such protease or the

<img file="MX337729B_D0052.tif" />

protease fragment is capable of cleaving a protein from the exocytic fusion apparatus of a nociceptive sensory afferent;

b. the second strand encompasses the TM and the translocation domain that is capable of displacing the protease or protease fragment within an endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent; and the first and second chains are disulfide linked.

During use, the single chain or double chain polypeptide of the invention treats, prevents, or improves pain.

During use, a therapeutically effective amount of a single chain or double chain polypeptide of the invention is administered to a patient.

The present invention addresses a wide range of pain conditions, particularly chronic pain conditions. Preferred conditions include cancerous and non-cancerous pain, inflammatory pain, or neuropathic pain. The opiates of the present application are particularly suitable for treating inflammatory pain, although they are less suitable for treating neuropathic pain. Galanin tablets are appropriate for treating neuropathic pain.

During use, the polypeptides of the present invention are typically employed in the form of a pharmaceutical composition in association with a pharmaceutical carrier, diluent, and / or excipient, and

INSTITL

Dt í Pl although the exact shape of the composition can be adapted to the mocTcO ^ of administration. Administration is preferably to a mammal, preferably a human.

The polypeptides can for example be used in the form of a sterile solution for intra-articular administration or intracranial administration. Spinal injection (eg epidural or intrathecal) is preferred.

The dosage ranges for administration of the polypeptides of the present invention are those that produce the desired therapeutic effect. It will be appreciated that the required dosage range depends on the exact nature of the components, the route of administration, the nature of the formulation, the age of the patient, the nature, the degree, or the severity of the condition. of the patient, contraindications, if any, and the judgment of the attending physician.

Convenient daily dosages are in the range of 0.0001-1 mg / kg, preferably 0.0001 - 0.5 mg / kg, more preferably 0.002-0.5 mg / kg, mg / kg of and particularly preferred 0.004 - 0.5. The dosage unit may vary from less than 1 microgram to 30mg, but will typically be in the region of 0.01 to 1mg per dose, which can be administered daily or preferably less frequently, for example weekly or semi-annually.

A particularly preferred dosage regimen is based on 2.5 ng of the fusion protein (eg CPNv / A) as the

<img file="MX337729B_D0053.tif" />

1X dose. In this regard, preferred dosages are in the range of 1X-100X (i.e. 2.5-250 ng). This dosage range is significantly lower (ie at least ten times, typically one hundred times less) than would be employed with other types of analgesic molecules such as NSASEQ ID S, morphine, and gabapentin. On the other hand, the aforementioned difference is magnified considerably when the same comparison is made on a molar basis - this is because the fusion proteins of the present invention have a considerably higher molecular weight than the "small" molecule therapeutics.

However, wide variations in the required dosage should be expected, depending on the exact nature of the components, and the differentiation efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Convenient compositions for injection may be in the form of solutions, suspensions or emulsions, or dry powders that are dissolved or suspended in a convenient vehicle prior to use.

Liquid unit dosage forms are typically prepared using a sterile pyrogen-free vehicle. The active ingredients, depending on the concentration and the vehicle used, can be dissolved or suspended in the vehicle.

In preparing administrable solutions,

IM

INST.'WTC C-. L ".

<img file="MX337729B_D0054.tif" />

Polypeptides can be dissolved in a vehicle, the solution being made isotonic if necessary by the addition of sodium chloride and sterilized by filtration through a sterile filter using aseptic techniques before filling into convenient sterile vials or ampoules and selalr. Alternatively, if the stability of the solution is adequate, the solution in its sealed containers can be autoclaved.

Advantageously additives such as buffering, solubilizing, stabilizing, preservative or bactericidal, suspending or emulsifying agents can be dissolved in the vehicle.

Dry powders that are dissolved or suspended in a convenient vehicle prior to use can be prepared by filling the pre-sterilized pharmaceutical substance and other ingredients in a sterile container using aseptic techniques in a sterile area.

Alternatively the polypeptides and that other ingredients can be dissolved in an aqueous vehicle, the solution is filter sterilized and distributed in convenient containers using aseptic techniques in a sterile area. The product is then lyophilized, dried and the containers are aseptically sealed.

Convenient parenteral suspensions for intramuscular, subcutaneous, or intradermal injection are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, rather than dissolved, and sterilization cannot be accomplished by filtration. The

IΜ

<img file="MX337729B_D0055.tif" />

INSTITUTE ΜέΧ'Ο.ΑΝΟ Of the ΡΚΟΟΌ'ΛΟ INOUSTf.iAL components can be isolated in a sterile state or alternatively sterilized after isolation, for example by gamma radiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition (s) to facilitate uniform distribution of the components.

Definitions section

The target portion (TM) means any chemical structure associated with an agent that functionally inactivates a binding point to cause a physical association between the agent and the surface of a target cell. In the context of the present invention, the target cell is a nociceptive sensory afferent. The term TM includes any molecule (i.e. a natural molecule, or a chemically / physically modified variant) that is capable of binding to a binding point in the target cell, such binding point is capable of internalization (eg endosome formation ) - also referred to as receptor mediated endocytosis. TM may possess an endosomal function of membrane translocation, in this case the separate components of TM and the translocation domain need not be present in an agent of the present invention.

TM of the present invention binds (preferably specifically binds) to a nociceptive sensory afferent (eg, a primary nociceptive afferent). In this regard, "binding specifically" means that TM binds to a sensory afferent

IMPI

INSTITUTE λ'ΟΟΟΑΝΟ EU LA 12 V^{: rr}INcuoi

<img file="MX337729B_D0056.tif" />

nociceptive (eg, a primary nociceptive afferent) with a higher affinity that binds to other neurons such as non-nociceptive afferents, and / or to motor neurons (ie the natural target for the neurotoxin clostridial holotoxin). The term "specific binding" can also mean that a given TM binds to a given receptor, for example the ORL ^ receptor with a binding affinity (Ka) of 10®M '¹ or greater, preferably 10⁷ M ¹ or greater, preferably 10⁸ M ~¹ or greater, and more preferably, 1 O⁹ M '¹ or older.

For the purposes of this invention, an agonist is defined as a molecule that is capable of stimulating the exoclastic fusion process in a target cell, a process that is susceptible to inhibition by a protease (or a fragment thereof) capable of breaking a protein from the exocytic fusion apparatus in the target cell.

Accordingly, the particular definition of agonist of the present invention would exclude many molecules that would be conventionally considered to be agonists.

For example, nerve growth factor (NGF) is an agonist in terms of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when determined by the above criteria because it is not a major inducer of exocytic fusion. Furthermore, the process that NGF stimulates (i.e. cell differentiation) is not susceptible to inhibition by

<img file="MX337729B_D0057.tif" />

protease activity of a non-cytotoxic toxin molecule.

The term "fragment when used in relation to a protein" means a peptide having at least thirty-five, preferably at least twenty-five, more preferably at least twenty, and even more preferably at least ten amino acid residues of the protein in question.

The term "variant when used in relation to a protein" means a peptide or a protein peptide fragment containing one or more amino acid analogues (eg, an artificial amino acid), or a substituted coupling.

The term "derivative, when used in relation to a protein, means a protein that encompasses the protein in question, and another sequence of the peptide. The additional peptide sequence should preferably not interfere with the basic folding and thus with the conformational structure of the original protein. Two or more peptides (or the fragments, or variants) can be brought together to form a derivative. Alternatively, a peptide (or fragment, or variant) can bind to an unrelated molecule (eg, a second, unrelated peptide). Derivatives can be chemically synthesized, but will typically be prepared by recombinant nucleic acid methods. Additional components such as lipids, and / or polysaccharides, and / or polyketide components can be included.

Throughout this specification, the reference to the Receiver

ENT ^ includes all members of the receptor family

<img file="MX337729B_D0058.tif" />

ORLv Members of the ORLi receptor family typically have a seven transmembrane domain structure and bind to proteins of the Gt and G families_or. A method for determining the stimulating activity of the G protein of ORLt receptor ligands is given in Example 12. A method for measuring the reduction in cellular cAMP levels after ORL activation! It is given in Example 11. Another characteristic of members of the ORLt receptor family is that they can typically bind to nociceptin (the natural ligand of ORLJ. As an example, all alternative ORLt receptor splice variants are members of the ORL receptor! Family.

The term non-cytotoxic means that the protease molecule in question does not kill the target cell to which it has been redirected.

The protease of the present invention includes all unnatural cytotoxic proteases that are capable of breaking one or more proteins from the exocytic apparatus of fusion in eukaryotic cells.

The protease of the present invention is preferably a bacterial protease (or a fragment thereof). Preferably the bacterial protease is selected from the genera Clostridium or Neisseria (for example a Clostridial L chain, or an IgA neisserial protease preferably from N. gonorrhoeae).

The present invention also includes modified nocytotoxic proteases, which include amino acid sequences.

J KA P

INDUS-nUAL that do not occur in nature and / or synthetic amino acid residues, as long as the modified proteases still show the aforementioned protease activity.

The protease of the present invention preferably demonstrates serine or metalloprotease activity (eg, endopeptidase activity). The protease is preferably specific for a SNARE protein (eg SNAP-25, slnaptobrevin / VAMP, or syntaxin).

Particular mention is made of the neurotoxin protease domains, for example the bacterial neurotoxin protease domains. Thus, the present invention includes the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of natural neurotoxins.

Exemplary neurotoxins are produced by Clostridia, and the term clostridial neurotoxin includes neurotoxins produced by C. tetan! C. (TeNT) 1 and AG serotypes of C. botulinum (BoNT), as well as the closely related BoNT-like neurotoxins produced by C. baratii and C butyricum. The aforementioned abbreviations are used throughout the present description. For example, the BoNT / A nomenclature denotes the source of neurotoxin as BoNT (serotype A). The corresponding nomenclature applies to other BoNT serotypes.

The terminal L chain fragment means a component of the L chain of a neurotoxin, a fragment demonstrating a

<img file="MX337729B_D0059.tif" />

activity and metalloprotease and is capable of proteolytically breaking a vesicle and / or a protein associated with the plasma membrane involved in cellular exocytosis.

A translocation domain is a molecule that allows translocation of a protease (or a fragment thereof) into a target cell such that functional expression of protease activity occurs within the cytosol of the target cell. Whether any molecule (eg, a protein or a peptide) possesses the indispensable translocation function of the present invention can be confirmed by a number of conventional assays.

For example, Shone C. (1987) describes an in vitro analysis that uses liposomes, which are attacked with a test molecule. The presence of the indispensable translocation function is confirmed by the release of the K + and / or NAD-labeled liposomes, which can be easily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167 (1): p. 175-180],

Another example is provided by Blaustein R. (1987), who describes a simple in vitro analysis using flat two-layer phospholipid membranes. The membranes are attacked with a test molecule that the indispensable function of translocation is confirmed by an increase in conductance across the membranes [see Blaustein (1987) FEBS Letts; vol. 226, No. 1: p. 115-120].

Additional methodology to allow determination of fusion and membrane identification of ϊ * · JA domains

Ί ·. . · Γ l \ si; tí

Ll LA Γ?.; '·! ·:

L.LLU translocation suitable for use in the present invention is thus provided by Methods in Enzymology Vol 220 and 221, Membrane Techniques, parts A and B, Academic Press 1993.

The translocation domain is preferably capable of ion-permeable pore formation in lipid membranes under low pH conditions. It has been found preferable to use only those portions of the protein molecule capable of forming pores within the endosomal membrane.

The translocation domain can be obtained from a microbial source of protein, particularly from a bacterial or viral source of protein. Therefore, in one embodiment, the translocation domain is a translocation domain of an enzyme, such as a bacterial toxin or a viral protein.

It is well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of the virally expressed proteins of the membrane fusion are capable of forming such pores. Such domains can be used in the present invention.

The translocation domain may be of clostridial origin, namely domain H_N (or a functional component thereof). H_Nmeans a portion or fragment of the H chain of a clostridial neurotoxin approximately equivalent to half of the amino terminus of the H chain, or the domain that corresponds to that fragment in the intact H chain. It is preferred that the chain H • _k

MLX'C INSTITUTE

Dt La r

INDUST substantially lacks the natural binding function of component H_c of the chain H. In this respect, the function H_c can be removed by canceling the amino acid sequence of H_c (either at the DNA synthesis level, or at the post-synthesis level by means of nuclease or protease treatment). Alternatively, the Hc function can be inactivated by chemical or biological treatment. Thus, the H chain is preferably unable to bind to the binding point in a target cell to which the native clostridial neurotoxin (i.e. holotoxin) binds.

In one embodiment, the translocation domain is an H domain_n (or a fragment thereof) of a clostridial neurotoxin. Examples of suitable clostridial translocation domains include:

Botulinum neurotoxin type A - amino acid residues (449-871)

Botulinum neurotoxin type B - amino acid residues (441-858) Botulinum neurotoxin type C - amino acid residues (442-866) Botulinum neurotoxin type D - amino acid residues (446-862) Botulinum neurotoxin type E - amino acid residues (423- 845) Botulinum neurotoxin type F - amino acid residues (440-864) Botulinum neurotoxin type G - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879).

For further details on the genetic basis of toxin production in Clostridium botulinum and C tetani., We refer to Romperson et al. (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_N Includes natural portions of H_N of the neurotoxin and the modified portions of H_N having amino acid sequences that do not occur in nature and / or synthetic amino acid residues, as long as the modified H portions_n they still show the translocation function.

Alternatively, the translocation domain may have a non-clostridial origin (see figure 4). Examples of the noclostridial origins of the translocation domain include, but are not restricted to, the translocation domain of diphtheria toxin [O = Keefe et al., Proc. Nati. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], the translocation domain of Pseudomonas type A exotoxins [Prior et al. Biochemistry (1992) 31, 3555-3559], the anthrax toxin translocation domains [Blanke et al. Proc. Nati. Acad. Sci. USA (1996) 93, 8437-8442], a variety of translocation function hydrophobic or hydrophobic peptides [Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp.1986- 1992], The translocation domain may reflect the translocation domain present in a natural protein, or may include variations of the amino acid as long as the variations do not destroy the translocation ability of the translocation domain.

The particular examples of the viral domains of

<img file="MX337729B_D0060.tif" />

Convenient translocations for use in the present invention include certain translocation domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation function (i.e. membrane fusion and vesiculation) of a number of fusogenic amphiphilic peptides derived from the hemagglutinin N-terminal region of the influenza virus. Other virally expressed membrane fusion proteins known to have the desired translocation activity are a translocation domain of a Semliki Forest virus (SFV) fusogenic peptide, a G glycoprotein translocation domain of the virus translocation domain of vesicular stomatitis (VSV), a translocation domain of the SER virus F protein, and a translocation domain of the Foamy virus envelope glycoprotein. Virally encoded Aspike proteins have particular use in the context of the present invention, eg, the SFV E1 protein and the VSV G protein G protein.

The use of the translocation domains indicated in the table (below) includes the use of the variants of their sequences. A variant may encompass one or more nucleic acid conservative substitutions and / or nucleic acid deletions or insertions, provided that the variant possesses the requisite translocation function. A variant may also encompass one or more amino acid substitutions and / or amino acid deletions or insertions, provided the variant possesses the requisite displacement function.

<img file="MX337729B_D0061.tif" />

Of the

INDUSTRIAL

<img file="MX337729B_D0062.tif" />

<td>Domain Source translocation</td><td>Waste amino acids</td><td>References</td>

<td>Diphtheria toxin</td><td> 194-380</td><td>Silverman et al, 1994, J. Biol. Chem. 269,22524-22532 London E., 1992, Bíochem. Biophys. Acta., 1113, 25-51</td>

<td>Domain II of Pseudomonas exotoxins</td><td> 405-613</td><td>Prior ei ai, 1992, Biochemistry 31, 3555-3559 Kíhara &amp; Pastan, 1994, Bioconj Chem. 5, 532-538</td>

<td>Hemagglutinin from influenza virus</td><td>GLFGAIAGFOGWE GMIDGWYG, and its variants</td><td>Plañir etal, 1994, J. Biol. Chem, 269, 12918-12924 Wagner et a!., 1992, PNAS, 89, 7934-7938 Murata et al, 1992, Biochemistry 31, 1986-1992</td>

<td>Semliki Forest virus fusogenic protein</td><td>Translocation domain</td><td>Kietian et al, 1996, J Cell Biol. 134 (4), 863-872</td>

<td>G glycoprotein vesicular stomatitis virus</td><td> 118-139</td><td>Yao et al, 2003, Virology 310 (2), 319-332</td>

<td>virus protein Fde SER</td><td>Translocation domain</td><td>Seth eí al, 2003, J Viral 77 (11) 6520-6527</td>

<td>Foamy virus envelope glycoprotein</td><td>Translocation domain</td><td>Picard-Maureau et al, 2003, J Viral. 77 (8), 4722-4730</td>

Br v Description of the Drawing

<img file="MX337729B_D0063.tif" />

Figures

Figure 1 Purification of an LC / A-nociceptin H fusion protein_n/TO

Figure 2 Purification of a nociceptin LC / A-hn / A fusion protein

Figure 3 Purification of an LC / C-nociceptin H fusion protein_n/ C

Figure 4 Purification of the LC / A-met enkephalin-HN / A fusion protein

Figure 5 Comparison of the binding efficiency of an LC / A-nociceptin-HN / A fusion protein and a nociceptin-LC / A-HN / A fusion protein

Figure 6 In vitro catalytic activity of an LC / A-nociceptin-HN / A fusion protein

Figure 7 Purification of an LC / A-nociceptin variant-HN / A fusion protein

Figure 8 Comparison of the binding efficiency of an LC / A-nociceptin-HN / A fusion protein and a variant LC / A-nociceptin NH / A fusion protein

Figure 9 The LC / A-nociceptin NH / A fusion protein family expressed / purified with the variable length spacer products

Figure 10 Inhibition of SNAP-25 SP cleavage and cleavage by CPN-A

Figure 11 Inhibition of SNAP-25 cleavage and cleavage of SP

IMPI institute Λ · ρ ·: ο

CO LA O..UUU

IND ^ uUL ___ for very long periods of time after exposure

<img file="MX337729B_D0064.tif" />

from DRG to CPN-A

Figure 12 SNAP-25 breakdown by CPNv-A

Figure 13 SNAP-25 cleavage for very long periods of time after DRG exposure to CPNv-A Figure 14 Fusion mediated displacement of [3H] -nociceptin binding of CPNv-A

Figure 15 expressed / purified CPNv (Ek) product

Figure 16 SNAP-25 breakdown by CPNv (Ek) -A

Figure 17 expressed / purified product of CPNv-C

Figure 18 Syntaxin cleavage of CPNv-C

Figure 19 Efficacy of CPN-A in the mechanical acute capsicin-induced mechanical allodynia model

Figure 20 Efficacy of CPN-A in the model of etreptozotocin-induced peripheral diabetic neuropathy (STZ) - peripheral Figure 21 efficacy of CPNv-A in the model of mechanical allodynia induced by acute capsicin

Figure 22 LC / A-CPLE-H product_N/ A expressed / purified

Figure 23 LC / A-CPBE-H product_N/ A expressed / purified

Figure 24 CPOP-A product expressed / purified

Figure 25 expressed / purified CPOPv-A product

Figure 26 In Vitro Breakdown of SNPA-25 in a DRG Cell Model

Figure 27 expressed / purified CPNv-A-FXa-HT (his tag detached)

Figure 28 In vitro efficacy of LC / A68 fusion proteins.

<img file="MX337729B_D0065.tif" />

nociceptin-HN / A with variable spacer length, determined by the ligand competition assay

Figure 29 In Vitro Efficacy of LC / Anociceptin-HN / A Fusion Proteins with Variable Spacer Length Determined by In Vitro Breakdown of SNAP-25

Detailed description of the invention

The figures will now be described in more detail.

Figure 1 - Purification of an LC / Anociceptin-HN / A fusion protein

Using the methodology outlined in Example 9, an LC / A-nociceptin-HN / A fusion protein was purified from Escherichia Coli BL21 cells. Briefly, the soluble products obtained after cell destruction were applied to a nickel loaded affinity capture column. The bound proteins were eluted with 100mM imidazole, treated with factor XA to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied to a second nickel-loaded affinity capture column. Samples from the purification procedure were determined by SDS-PAGE (panel A) and Western blotting (panel B). Anti-nociceptin antisera (obtained from Abcam) were used as primary antibodies for Western blotting. The final purified material in the presence and absence of reducing agent is identified in the lanes marked with [-] and [+] respectively.

Figure 2 - Purification of a nocic ptina69 fusion protein

<img file="MX337729B_D0066.tif" />

lc / ah_n/to

Using the methodology described in Example 9, a nociceptin-LC / A-HN / A fusion protein was purified from Escherichia Coli BL21 cells. Briefly, the soluble products obtained after cell destruction were applied to a nickel loaded affinity capture column. The ligated proteins were rinsed with 100 mM imidazole, treated with factor XA to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied to a second nickel-loaded affinity capture column. Samples from the purification procedure were determined by SDS-PAGE (panel A) and Western blotting (panel B). Anti-nociceptin antisera (obtained from Abcam) were used as primary antibodies for Western blotting. The final purified material in the presence and absence of reducing agent is identified in the lanes marked with [-] and [+] respectively.

Figure 3 - Purification of an LC / Cnociceptin-H fusion protein_N/ C

Using the methodology described in Example 9, an LC / C-nociceptin-H fusion protein_N/ C was purified from Escherichia Coli BL21 cells. Briefly, the soluble products obtained after cell destruction were applied to a nickel loaded affinity capture column. Bound proteins were rinsed with 100mM imidazole, treated with factor XA to activate the fusion protein and remove the tag

<img file="MX337729B_D0067.tif" />

of maltose binding protein (MBP), then reapplied to a second nickel-loaded affinity capture column samples from the purification procedure were determined by SDS-PAGE (panel A) and Western blotting (panel B). Anti-nociceptin antisera (obtained from Abcam) were used as primary antibodies for Western blotting. The final purified material in the presence and absence of reducing agent is identified in the lanes marked with [-] and [+] respectively.

Figure 4 - Purification of an LC / A-met eencepha I i na-H fusion protein _N/TO

Using the methodology described in Example 9, an LC / A-met enkephalin-H fusion protein_N/ A was purified from Escherichia Coli BL21 cells. Briefly, the soluble products obtained after cell destruction were applied to a nickel loaded affinity capture column. The ligated proteins were rinsed with 100 mM imidazole, treated with factor XA to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied to a second nickel-loaded affinity capture column. Samples from the purification procedure were determined by SDS-PAGE. The final purified material in the presence and absence of reducing agent is identified in the lanes marked with [-] and [+] respectively.

Figure 5 - Comparison of the efficiency of nociceptin-LC / AH fusion protein binding_N/ A and a protion of fuion d

<img file="MX337729B_D0068.tif" />

nocic ptina-LC / AH_N/TO

The ability of nociceptin es to bind to the ORL receptor was determined.₁ using a simple test based on competition. The primary dorsal root ganglia (DRG) primitives were exposed to various concentrations of test material in the presence of 1nM [3H] -nociceptin. The reduction in the specific binding of the radiolabelled ligand was determined by scintillation counting, and plotted in comparison to the efficacy of the unlabelled ligand (nociceptin Tocris). It is clear that the LC / Anociceptin-HN / A fusion was far superior to the nociceptin-LC / AHN ​​/ A fusion when interacting with the ORLj receptor.

Figure 6 In vitro catalytic activity of an LC / A-nociceptin-HN / A fusion protein

The in vitro endopeptidase activity of the LC / A-nociceptin-HN / A fusion protein was determined essentially as described in Chaddock et al 2002, Prot. Express Purif. 25, 219-228. Briefly, the SNAP-25 peptides immobilized in the ELISA plate were exposed to different concentrations of fusion protein for 1 hour at 37 ° C. After a series of washes, the amount of SNPA-25 peptide cleaved was quantified by reactivity with specific antisera.

Figure 7 Purification of a variant-HN / A fusion protein from

LC / A-nociceptin

Using the methodology described in Example 9, an LC / A-nociceptin variant-HN / A fusion protein was purified from Escherichia Coli BL21 cells.

<img file="MX337729B_D0069.tif" />

Briefly, the soluble products obtained after cell destruction were applied to a nickel loaded affinity capture column. The ligated proteins were rinsed with 100 mM imidazole, treated with factor Xa to activate the fusion protein and remove the maltose binding protein (MBP) tag, then reapplied to a second nickel-loaded affinity capture column. Samples from the purification procedure were determined by SDS-PAGE. The final purified material in the presence and absence of reducing agent is identified in the lanes marked with [-] and [+] respectively.

Figure 8 Comparison of the binding efficiency of an LC / A-nociceptin-HN / A fusion protein and a variant LC / A-nociceptin NH / A fusion protein

The ability of nociceptin es to bind to the ORL receptor was determined.₁ using a simple test based on competition. Primary dorsal root ganglia (DRG) cultures were exposed to various concentrations of test material in the presence of 1nM [3H] -nociceptin. The reduction in the specific binding of the radiolabelled ligand was determined by scintillation counting, and plotted in comparison to the efficacy of the unlabelled ligand (nociceptin Tocris). It is clear that the LC / Anociceptin-HN / A fusion was far superior to the LC / A-nociceptin variant-HN / A fusion interacting with the ENT receptor.

Figure 9 The family of the LC / A-nocic ptina73 fusion protein

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HN / A Expressed / Purified With Variable Spacer Products

Using the methodology described in Example 9, the LC / A-CPN-HN / A fusion variants consisting of GS10, GS30 and HX27 are purified from E. coli cell paste. Samples of the LC / A-CPN (GS10) -H purification_N/ A, LC / A-CPN (GS15) -H_N/ A, LC / A-CPN (GS25) -H_n/ A, LC / A-CPN (GS30) -H_n/ A and LC / A-CPN (HX27) H_n/ A are determined by SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-chain species with the expected molecular mass of CPBE-A. Top panel: standard test of molecular mass markers; S - total soluble fraction of E. coli protein; FT = proteins that do not bind to the N¡-loaded sepharose column²⁺; = fusion protein eluted by the addition of imidazola.

Lower panel: lane 1 = standard test of molecular mass markers; lane 2 = total soluble fraction of E. coli protein; lane 3 = material purified after initial capture in N¡-loaded sepharose²⁺; lane 4 = factor Xa-treated material before initial capture in N-loaded sepharose²⁺; lane 5 = final material purified after activation with factor Xa (5 pl); lane 6 = final material purified after activation with factor Xa (10 pl); lane 7 = final material purified after activation with factor Xa (20 µΙ); lane 8 = final material purified after activation with factor Xa + DTT (5 pl); lane 9 = final material purified after activation with factor Xa +

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FROM THE PROFIF.DAB Go ».- ·&gt;

INDUSTRIAL '«SeAlHÍDTT (10 pl); lane 10 = final material purified after activation with factor Xa + DTT (20 pl).

Figure 10 Inhibition of SNAP25 cleavage and SP release by CPN-A

Briefly, primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPN-A for 24 hours. Cellular proteins were separated by SDS-PAGE, subjected to Western blott tests and sampled with anti-SNPA25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by densitometric analysis and plotted against the fusion concentration (dotted line). The material was also recovered for a substance P content analysis using specific EIA equipment. Inhibition of substance P release is illustrated by solid lines. The melt concentration required to achieve a maximum SNAP-25 breakdown of 50% is estimated to be at

6.30 + 2.48 nM.

Figure 11 Inhibition of SNAP25 cleavage and cleavage of SP for very long periods of time after exposure of DRG to CPN-A

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT / A) was used as a control.

After the initial exposure, the extracellular material was removed by washing and the cells were incubated at 37 ° C for

IMPI

MEXICAN INSTITUTE '

OF PROPERTY ..... Ά

INDUSTRIAL ~^J -LL Different periods of time. At specific time points, cellular proteins were separated by SDS-PGE, Western blott assayed, and sampled with anti-SNPA-25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by densitometric analysis and plotted against the fusion concentration (dotted line). The material was also recovered for a substance P content analysis using specific EIA equipment. Inhibition of the release of substance P is illustrated with the solid line.

Figure 12 SNAP-25 breakdown by CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPNv-A for 24 hours. Cellular proteins were separated by SDS-PGE, subjected to Western blott tests and sampled with anti-SNPA-25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by means of densitometric analysis. The fusion concentration required to achieve a maximum SNAP-25 breakdown of 50% is estimated to be 1.38 +0.36 nM. Figure 13 SNAP-25 cleavage over very long periods of time after DRG exposure to CPNv-A

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPNv-A for 24 hours.

Botulinum neurotoxin (BoNT / A) was used as a control.

After the initial exposure, the extracellular material was removed by washing and the cells were incubated at 37 ° C for

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<img file="MX337729B_D0073.tif" />

different periods of time. At specific time points, cellular proteins were separated by SDS-PGE, Western blott assayed, and sampled with anti-SNPA-25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by means of densitometric analysis.

Figure 14 Fusion-mediated displacement of CPNv-A from the [3H] -nociceptin bond

The ability of nociceptin es to bind to the ORL receptor was determined using a simple competition-based assay. Primary dorsal root ganglia (DRG) cultures were exposed to various concentrations of test material in the presence of 1nM [3H] -nociceptin. The reduction in the specific binding of the radiolabelled ligand was determined by scintillation counting, and plotted in comparison to the efficacy of the unlabelled ligand (nociceptin Tocris). It is clear that the LC / A-nociceptin variant NH / A fusion was superior to the LC / Anociceptin-H fusion._N/ A (labeled CPN-LHnA) when interacting with the ORL receptor ^

Figure 15 expressed / purified CPNv (Ek) product

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-chain species of the expected molecular mass of CPNv (Ek). Lane 1 = standard test of molecular mass markers; lane 2 = total soluble fraction of E. coli protein; lane 3 = purified material after

<img file="MX337729B_D0074.tif" />

initial catch in sepharose loaded with Ni²⁺; lane 4 = final material purified after activation with enterokinase (5 pl); lane 5 = final material purified after activation with enterokinase (10 pl); lane 6 - final material purified after activation with enterokinase (20 pl); lane 7 = final material purified after activation with enterokinase + DTT (5 pl); lane 8 = final material purified after activation with enterokinase + DTT (10 pl); lane 9 = final material purified after activation with enterokinase + DTT (20 pl).

Figure 16 SNAP-25 breakdown by CPNv (Ek) -A

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPNv-A (Ek) -A for 24 hours. Cellular proteins were separated by SDS-PGE, Western blott assayed, and sampled with anti-SNPA25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by means of densitometric analysis. CPNv-A as prepared in Example 9 was used for comparison purposes. The percentage breaks in SNAP-25 are illustrated by means of CPNv (Ek) -A (labeled En activated) and CPNv-A (labeled Xa activated).

Figure 17 - expressed / purified CPNv-C product

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-chain species of the expected molecular mass of Cpnv.-C. Lane 1 = benchmark

<img file="MX337729B_D0075.tif" />

molecular mass markers; lane 2 = total soluble protein fraction of 'E. coli; lane 3 = purified material after initial capture in Ni-loaded sepharose²⁺; lane 4 = factor Xa treated material before final capture in Ni-loaded sepharose²⁺; lane 5 = purified material after Ig second capture in N-loaded sepharose²⁺; 6 = purified final material; lane 7 - purified final material + DTT; lane 8 = benchmark molecular mass markers.

Figure 18 Syntaxin cleavage of CPNv-C

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPNv-C for 24 hours. Cellular proteins were separated by SDS-PGE, subjected to Western blott tests, and sampled with anti-syntaxin to facilitate determination of cleavage with syntaxin. The percentage of broken syntaxin was calculated by means of densitometric analysis. The melt concentration required to achieve a maximum syntaxin breakdown of 50% is estimated to be 3.13 ± 1.96 nM. Figure 19 Efficacy of CPN-A in the mechanical acute capsicin-induced mechanical allodynia model

LC / A-nociceptin-H fusion ability_N/ A (CPN / A) to inhibit capsaicin-induced mechanical allodynia was evaluated after subcutaneous intraplantar injection into the hind paw of the rat. Test animals were evaluated for I leg paw withdrawal frequency (PWF%) in response to a series of stimuli with 10 g of Von Frey filaments (10 stimuli x 3

<img file="MX337729B_D0076.tif" />

tests) before recruiting the study (pre-treatment); after interaplantar treatment with CPN / A but before capsaicin (Pre-CAP) and after attack with capsaicin after injection of CPN / A (by means of responses at 15 'and 30'; CAP). Capsin attack was accomplished by injecting 10 pL of a 0.3% solution. Sample dilutions were prepared in

0.5BSA / saline.

Figure 20 - Efficacy of NPC-A in the model of etreptozotocin-induced peripheral diabetic neuropathy (STZ) (neuropathic pain)

Male DSprague-Sawley rats (250-300 g) are treated with 65 m / kg STZ in citrate buffer (iv) and glucose and blood lipids were measured weekly to define model adequacy. The paw withdrawal threshold (PWT) is measured in response to a series of stimuli with Von Frey filaments during a type period. Allodynia is said to have been established when the PWT on two consecutive test days (1 week apart) measures less than 6g on the scale. At this point rats are randomly assigned to either a saline group (negative efficacy control), a gabapentin group (positive efficacy control), or a test group (NPC / A). The test materials (20-25 μΙ) are injected subcutaneously as a single injection except gabapentin) and the PWT is measured 1 day after testing and periodically after a period of more than 2 weeks. Gabapentin (30 mg / kg i, p. At 3 ml / kg volume of

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Figure 21 efficacy of CPNv-A in the model of mechanical allodynia induced by acute capsicin

Ability of LC / A-variant nociceptin H fusion_n/ A (CPNv / A) to inhibit capsaicin-induced mechanical allodynia was evaluated after subcutaneous intraplantar injection into the hind paw of the rat. Test animals were evaluated for I leg removal frequency (PWF%) in response to a series of stimuli with 10 g of Von Frey filaments (10 stimuli x 3 tests) before recruiting the study (pre- treatment); after interaplantar treatment with vNCP / A but before capsaicin (Pre-CAP) and after attack with capsaicin after injection of vNPC / A (by means of responses at 15 'and 30'; CAP). Capsin attack was accomplished by injecting 10 pL of a 0.3% solution. Sample dilutions sep repaired at 0.5BSA / saline. These data are expressed as a normalized paw withdrawal frequency differential at which the difference between the peak response (postcapsicin) and the baseline response is expressed as a percentage. With this analysis it can be seen that Cpnv./A is more potent than CPN / A since lower doses of Cpnv./A are required to achieve a similar analgesic effect than that observed with

CPN / A.

Figure 22 product of LC / A-CPLE-H_N/ Squeezed / purified

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<img file="MX337729B_D0077.tif" />

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-stranded species of the expected molecular mass of CPLE-A. Lane 1 = standard test of molecular mass markers; lane 2 = total soluble fraction of E. coli protein; lane 3 = purified material after initial capture in Ni-loaded sepharose²⁺; lane 4 = factor Xa treated material before final capture in Ni-loaded sepharose²⁺; lane 5 = purified material after the second N-loaded sepharose capture²⁺ 6 = purified final material; lane 7 = purified final material + DTT;

Figure 23 LC / A-CPBE-H product_N/ A expressed / purified

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-chain species of the expected molecular mass of CPBE-A. Lane 1 = total soluble fraction of E. coli protein; lane 2 = purified material after initial capture in Ni-loaded sepharose²⁺; lane 3 = material treated with factor Xa before the final capture in sepharose loaded with N¡²⁺; lane 4: final material purified after activation with factor Xa (5 pl); lane 5 = final material purified after activation with factor Xa (10 pl); lane 6 = final material purified after activation with factor Xa (20 pl); lane 7 = final material purified after activation with factor Xa + DTT (5 pl); lane 8 = final material purified after

<img file="MX337729B_D0078.tif" />

activation with factor Xa + DTT (10 pl); lane 9 = final material purified after activation with factor Xa + DTT (20 µΙ), lane 10 = benchmark molecular mass markers.

Figure 24 CPOP-A product expressed / purified

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-stranded species of the expected molecular mass of CPOP-A. Lane 1 = benchmark molecular mass markers .; lane 2 = purified material after initial capture in Ni-loaded sepharose²⁺; lane 3 = factor Xa treated material before final capture in Ni-loaded sepharose²⁺; lane 4: purified material after the second Ni-loaded sepharose capture²⁺: lane 5 = final material purified after activation with factor Xa (5 µΙ); lane 6 = final material purified after activation with factor Xa (10 µΙ); lane 7 = final material purified after activation with factor Xa (20 µΙ); lane 8 = final material purified after activation with factor Xa + DTT (5 µΙ); lane 9 = final material purified after activation with factor Xa + DTT (10 µΙ); lane 10 = final material purified after activation with factor Xa + DTT (20 µΙ).

Figure 25 expressed / purified CPOPv-A product

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-chain species from the mass

IMPI

M EXICAN INSTITUTE OF The expected molecular INDUSTRIAL PRCPISDaO of CPOPv-A. Lane 1 = benchmark molecular mass markers .; lane 2 = total soluble fraction of E. coli protein; lane 3 = purified material after initial capture in Ni-loaded sepharose²⁺; lane 4 = factor Xa treated material before final capture in Ni-loaded sepharose²⁺; lane 5 = final material purified after activation with factor Xa (5 pl); lane 6 = final material purified after activation with factor Xa (10 pl); lane 7 = final material purified after activation with factor Xa (20 pl); lane 8 = final material purified after activation with factor Xa + DTT (5 pl); lane 9 = final material purified after activation with factor Xa + DTT (10 pl); lane 10 = final material purified after activation with factor Xa + DTT (20 pl). Figure 26 In Vitro Breakdown of SNPA-25 in a DRG Cell Model

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPOPv-A for 24 hours. Cellular proteins were separated by SDS-PGE, subjected to Western blott tests and sampled with anti-SNPA-25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by means of densitometric analysis.

Figure 27 expressed / purified CPNv-A-FXa-HT (his tag detached)

Proteins were subjected to SDS-PAGE before staining with Coomassie blue. The electrophoresis profile indicates the purification of a disulfide-linked double-chain species from the mass

<img file="MX337729B_D0079.tif" />

<img file="MX337729B_D0080.tif" />

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<img file="MX337729B_D0081.tif" />

expected molecular of CPNv-A-FXa-HT. Lane 1 = benchmark molecular mass markers .; lane 2 = total soluble fraction of E. coli protein; lane 3 = material treated with factor Xa before the final capture in sepharose loaded with N¡²⁺; lane 4 = final material purified after activation with factor Xa lane 5 = final material purified after activation with factor Xa + DTT

Figure 28 In vitro efficacy of LC / Anociceptin-HN / A fusion proteins with variable spacer length, determined by the ligand competition assay

The ability of the LC / A-nociceptin-HN / A es to bind to the ORLi receptor was determined using a simple competition-based assay. Primary dorsal root ganglia (DRG) cultures were exposed to various concentrations of test material in the presence of 1nM [3H] -nociceptin. The reduction in specific binding of the radiolabelled ligand was determined by scintillation counting, and plotted in comparison to the efficacy of the unlabelled ligand (nociceptin Tocris). The upper panel illustrates the displacement characteristics of the GSO, GS20, GS30 and Hx27 separators, while the lower panel illustrates the displacement obtained by the separate GS10, GS15 and GS25 fusion proteins. It is concluded that the GSO and GS30 spacers are ineffective, and that GS10 is ineffective, in displacing nociceptin from the ENT receptor ^

Figure 29 In vitro ficacy of the LC / A85 fusion proteins

<img file="MX337729B_D0082.tif" />

nocic ptina-HN / A with variable spacer length, determined by the in vitro cleavage of SNAP-25

Primary cultures of dorsal root ganglia (DRG) were exposed to various concentrations of CPN-A (with different spacer length) for 24 hours. Cellular proteins were separated by SDS-PGE, subjected to Western blott tests and sampled with anti-SNPA-25 to facilitate determination of SNP-25 disruption. The percentage of SNP-25 broken was calculated by means of densitometric analysis. The ineffective binding characteristics of the GS10 spaced fusion protein (see Figure 28) are reflected in the high fusion concentrations required to achieve intracellular SNPA-25 cleavage. Spaced GSO and GS30 fusion proteins were completely ineffective (date not shown). The GS15, 20 and spaced fusion proteins were similarly effective.

SEQ ID NOs

SEQ ID 1 LC / A DNA sequence

SEQ ID 2 H DNA sequence_N/ A SEQ ID 3 LC / B DNA Sequence

SEQ ID 4 H DNA sequence_N/ B

SEQ ID 5 LC / C DNA sequence

SEQ ID 6 H DNA sequence_N/ C

SEQ ID 7 DNA sequence of binder CPN-A SEQ ID 8 DNA sequence of binder A

SEQ ID 9 DNA sequence of the presentation insert of '-n de S?

INSTITUTO MEXICANO'Í 'DE LA PROPIEDAD v —INDUSTRIAL terminal nociceptin N SEQ ID 10 DNA sequence of ligand CPN-C SEQ ID 11 DNA sequence of ligand CPBE-A SEQ ID 12 DNA sequence of ligand CPNvar-A SEQ ID 13 DNA sequence of the LC / A-CPN-HN / A fusion SEQ ID 14 Protein sequence of the LC / A-CPN-HN / A fusion

SEQ ID 15 DNA sequence of N-LC / A-HN / A

SEQ ID 16 Protein sequence of the N-LC / AH fusion_W/TO

SEQ ID 17 DNA sequence of the LC / C-CPN-H fusion_N/ C

SEQ ID 18 Protein sequence of the LC / C-CPN-H fusion_N/ C

SEQ ID 19 DNA sequence of the LC / C-CPN-HN / C fusion (Linker) SEQ ID 20 Protein sequence of the LC / CCPN-H fusion_n/ C (A-binder)

SEQ ID 21 DNA sequence of the LC / A-CPME-H fusion_N/TO

SEQ ID 22 Protein sequence of the LC / A-CPME-H fusion_N/TO

SEQ ID 23 DNA sequence of the LC / A-CPBE-H fusion_N/TO

SEQ ID 24 Protein sequence of the LC / A-CPBE-H fusion_N/TO

SEQ ID 25 DNA sequence of the LC / A-CPNv-H fusion_n/TO

SEQ ID 26 Protein sequence of the LC / A-CPNv-H fusion_n/TO

SEQ ID 27 DNA sequence of the LC / A-CRN fusion [1-11J-HN / A

SEQ ID 28 Protein sequence of the LC / A-CPN [1-11] HN / A fusion

SEQ ID 29 DNA sequence of the LC / A-CPN fusion [[Y10] 1-11] HN / A SEQ ID 30 Protein sequence of the LC / ACPN fusion [[Y1 0] 1-11J-HN / A

<img file="MX337729B_D0083.tif" />

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SEQ ID 31 DNA sequence of the LC / A-CÍ fusion^s'N [fVl IJPTTfHN / A -——— --—

SEQ ID 32 Protein sequence of the LC / A-CPN fusion [[Y11] 111J-HN / A

SEQ ID 33 DNA sequence of the LC / A-CPN fusion [[Y14] 1-17] HN / A

SEQ ID 34 Protein sequence of the LC / A-CPN fusion [[Y14J1 17J-HN / A

SEQ ID 35 DNA sequence of the LC / A-CPN [1 -13J-HN / A fusion SEQ ID 36 Protein sequence of the LC / A-CPN [I -13] HN / A fusion

SEQ ID 37 CPN DNA sequence [1-17]

SEQ ID 38 CPN protein sequence [I -17]

SEQ ID 39 CPN DNA sequence [I -11]

SEQ ID 40 CPN protein sequence [I -11]

SEQ ID 41 CPN DNA sequence [[Y10] 1-11]

SEQ ID 42 CPN protein sequence [[Y10] 1-11] SEQ ID 43 CPN DNA sequence [[Y11] 1-11]

SEQ ID 44 CPN protein sequence [[Y 11] 1-11]

SEQ ID 45 CPN DNA sequence [[Y14] 1-17]

SEQ ID 46 NPC protein sequence [[Y14] 1-1 7] SEQ ID 47 NPC DNA sequence [I -13]

SEQ ID 48 NPC protein sequence [I -13]

SEQ ID 49 DNA sequence of CPNv (also known as

N [[R14K1 5] 1-17])

SEQ ID 50 Protein sequence of asN [[R14K1 5] 1-1 7])

CPNv

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<img file="MX337729B_D0085.tif" />

SEQ ID 51 Nociceptin-LC / AHi Spacer DNA Sequence_M/TO

SEQ ID 52 Protein sequence of the nociceptin -LC / A-Hi spacer fusion_M/TO

SEQ ID 53 DNA sequence of the CPN-A GS10 binder

SEQ ID 54 DNA sequence of the CPN-A GS15 binder

SEQ ID 55 DNA sequence of the binder CPN-A GS25

SEQ ID 56 DNA sequence of the binder CPN-A GS30

SEQ ID 57 DNA sequence of the CPN-A HX27 binder

SEQ ID 58 LC / A-CPN (GS 15) -H fusion DNA sequence_N/ A SEQ ID 59 Protein sequence of the LC / A-CPN (GS15) H fusion_n/TO

SEQ ID 60 LC / A-CPN (GS25) -H fusion DNA sequence_N/ A SEQ ID 61 Protein sequence of the LC / A-CPN (GS25) H fusion_n/ A SEQ ID 62 DNA sequence of activatable enterokinase binding CPNvar-A

SEQ ID 63 LC / A-CPNv (Ek) -H fusion DNA sequence_N/ A SEQ ID 64 Protein sequence of the LC / A-CPNv (Ek) H fusion_n/TO

SEQ ID 65 CPNvar-A Binder DNA Sequence SEQ ID 66 LC / C-CPNv-H Fusion DNA Sequence_n/ C (act. A) SEQ ID 67 Protein sequence of the LC / C-CPNv-H fusion_n/ C (act. A)

<img file="MX337729B_D0086.tif" />

SEQ ID 68 LC / A-CPLE-H fusion DNA sequence_N/ A SEQ ID 69 Protein sequence of the LC / A-CPLE-H fusion_N/ A SEQ ID 70 LC / A-CPOP-H fusion DNA sequence_N/ A SEQ ID 71 Protein sequence of the LC / A-CPOP-H fusion_N/ A SEQ ID 72 LC / A-CPOPv-H fusion DNA sequence_N/ A SEQ ID 73 Protein sequence of the LC / A-CPOPV-HN fusion / A SEQ ID 74 IgA protease DNA sequence

SEQ ID 75 lgA-CPNv-H fusion DNA sequence_N/ A SEQ ID 76 Protein sequence of the lgA-CPNv-H fusion_N/TO

SEQ ID 77 FXa-HT DNA sequence

SEQ ID 78 DNA sequence of CPNv-A-FXa-HT

SEQ ID 79 Protein sequence of the CPNv-A-FXa-HT fusion

SEQ ID 80 DT translocation domain DNA sequence

SEQ ID 81 DNA sequence of CPLE-DT-A

SEQ ID 82 Protein sequence of the CPLE-DT-A fusion

SEQ ID 83 TeNT LC DNA sequence

SEQ ID 84 DNA sequence of CPNv-TENT LC

SEQ ID 85 CPNV-TeNT LC fusion protein sequence SEQ ID 86 CPNvar-C ligand DNA sequence SEQ ID 87 LC / C-CPNv-H fusion DNA sequence_n/ C (act. C) SEQ ID 88 Protein sequence of the LC / C-CPNv-Hn ​​/ C (act. C) fusion

Examples

Example 1 - Preparation of clones of an LC / A foundation and

HN / A

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INDUSTRIAL

<img file="MX337729B_D0087.tif" />

The following procedure creates the LC and HN fragments for use as a building block for multi-domain fusion expression. This example is based on the preparation of a clone based on serotype A (SEQ ID 1 and SEQ ID 2), although the procedures and methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B ( SEQ ID 3 and SEQ ID 4) and serotype C (SEQ ID 5 and SEQ ID 6)]. Preparation of cloning and expression vectors pCR 4 (Invitrogen) is the chosen standard cloning vector, it is selected due to the lack of restriction sequences within the vector and the adjacent sequence primer sites for easy confirmation of construction. The expression vector is based on the pMAL expression vector (NEB), which has the desired sequences of I restriction within the multiple cloning site with the correct orientation for insertion of the construct (BamHI-Sa / l- PsfI- Η / πό111) A fragment of the expression vector has been removed to create a non-mobilizable plasmid and a variety of different fusion tags have been inserted to increase purification options.

Preparation of the protease insert (eg LC / A)

The LC / A (SEQ ID 1) is created in one of two ways:

The DNA sequence is designed by the back translation of the LC / A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (access point

<img file="MX337729B_D0088.tif" />

inst;

υ: ι. \ ι

BXA1_CLOBO) using one of a variety of software tools for reverse translation (eg EditSeq the best reverse translation for E. Coli (DNASTAR Inc.), or the Backtranslation v2.0 tool (Entelechon)). BamHI / Sa / l are incorporated at the 5 'and 3' ends respectively of the sequence, maintaining the correct reading frame. The DNA sequence is scanned (using software such as MapDraw, DNASTAR Inc.) for the restriction enzyme cleavage sequences incorporated during the back translation. Any break sequence that is found is common to those required by the cloning system is maintained and manually removed from the proposed coding sequence that ensures common use of the E. coli codon is maintained. The use of the E. codon Coli is determined by reference to computer programs such as a graphical codon usage analyzer (Geneart), and the content ratio% GC and codon usage of y determined by reference to published codon usage tables (eg. GenBank Issue 143, September 13, 2004). This optimized DNA sequence containing the LC / A open reading frame (ORF) is then synthesized commercially (eg by Entelechon, Geneart or SlgmaGenosys) and is provided in vector pCR4.

The alternative method is the use of PCR amplification of the existing DNA sequence with restriction enzyme sequences fiamHI and Sa / I incorporated into the 5 'and 3' PCR primers.

<img file="MX337729B_D0089.tif" />

<img file="MX337729B_D0090.tif" />

Ni-C '··· ..' ··

The complementary oligonucleotide primers are chemically synthesized by the supplier (for example MWGΓ or Sigma- · - 'Genosys), in such a way that each pair has the ability to hybridize to the opposite strips (end 3' that point towards each other) flanking the Clostridium target DNA extension, an oligonucleotide for each of the two DNA strips. To generate a PCT product, the pair of short oligoucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the “PCR Machine”) that can be changed the incubation temperature of the reaction tube automatically, changing between approximately 94 ° C (for denaturation),

55 ° C (for oligonucleotide fixation) and 72 ° (for synthesis).

Other reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase) each of the four blocks that make up the DNA dNTP nucleotide in equimolar amounts (50-200 µΜ) and an appropriate buffer for the enzyme optimized for Mg concentration²⁺ (0.5-5 mM).

The amplification product is cloned into pCR 4 using either TOPO TA cloning for Taq PCT products or Zero Blunt TOIPO for Pfu PCT products (both kits are commercially distributed by Invitrogen). The resulting clone is verified by sequencing. Any additional restriction sequences that are not compatible with the cloning system are then removed. ÍNi&gt; J '' • 'il. "-'SJ * · using site-directed mutagenesis (eg usbTTdo Quickchange (Stratagene Inc.)].

Preparation of the translocation insert (eg H_N)

The DNA sequence is designed by means of the back translation of the amino acid sequence H_N/ A [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (access point BXA1_CLOBO) using one of a variety of software tools for reverse translation (eg EditSeq the best reverse translation from E. Coli (DNASTAR Inc), or the Backtranslation v2.0 (Entelechon) tool.] A Pstl restriction sequence added to the N term and the Xbal Hindll stop codon at terminal C ensuring that the correct reading frame is maintained. The DNA sequence is analyzed (using software such as MapDraw, DNASTAR Inc.) to search for built-in restriction enzyme cleavage sequences during back translation. Any sequences found to be common to those required by the cloning system are manually removed from the common coding sequence ensuring that the use of the common E.coli elbow is maintained. E.coli codon usage is determined by reference to software programs such as the graphical codon usage analyzer (Geneart), and the% GC content and codon usage ratio of y determined by reference to published tables of codon usage (eg GenBank Issue 143, September 13, 2004). This optimized DNA sequence contains the LC / A open reading frame (ORF) is

<img file="MX337729B_D0091.tif" />

DE ί. /. Γ then

<img file="MX337729B_D0092.tif" />

L '/ iU r? JAL

<img file="MX337729B_D0093.tif" />

synthesized) commercially (for example by Entelechon, Geñe'arT ^ 'or ^ STg'mcP ^ - ··'

Genosys) and is provided in vector pCR4.

The alternative method is the use of PCR amplification of the existing DNA sequence with restriction enzyme Pst \ sequences and with the stop codon Xba \ Hindll incorporated in the 5 'and 3' PCR primers. PCT amplification is performed as described above. The PCR product is inserted into the pCR4 vector and verified by sequencing. Any additional restriction sequences that are not compatible with the cloning system are then removed using site-directed mutagenesis [eg using Quickchange (Stratagene Inc.)].

Example 2 - Preparation of an LC / Anociceptin-H fusion protein_NA (nociceptin is the N-terminus of the H chain_N) Preparation of the binder-nociceptin spacer insert

The LC-H binder_N it can be designed from the first principle using the existing binder sequence information as a template. For example the serotype A ligand (in this case defined as the inter-domain polypeptide region that exists between the cysteines of the disulfide bridge between LC and H_N) is 23 amino acids in length and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence it is understood that proteolytic activation in nature leads to an H domain_N that has an N terminal of the ALNDL sequence. This sequence information can be freely obtained from available database sources.

I

INSTiT

<img file="MX337729B_D0094.tif" />

such as GenBank (access number P10845) or Swissprot (access point BXA1_CLOBO). In this binder a factor Xa, nociceptin and separate site are incorporated; and using one of a variety of reverse translation software tools [eg EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the binding-ligand-spacer region. The restriction sites are incorporated into the DNA sequence and can be arranged as a BamHI-Sa / l site-nociceptinHhel-spacer-Spel-Psfl-Xbal-stop codon protease-H / ndIII (SEQ ID 7). It is important to ensure that the correct reading frame is maintained for the separator, nociceptin, and restriction sequences and that the Xbal sequence is not preceded by bases, TC, which would result in DAM mutilation. The DNA sequence is examined to find the incorporation of the restriction sequence, and any additional sequences are manually removed from the remaining sequence ensuring that the E.coll codon usage is maintained. The use of the E. codon coli is determined by reference to software programs such as the Graphical Codon Usage Analyzer (Geneart), and the% GC content and proportion of codon usage determined by reference to published codon usage tables (for example , GenBank issue 143, September 13, 2004). This optimized DNA sequence is then commercially synthesized (eg by Entelechon, Geneart or Sigma-Genosys) and is provided in vector pCR 4.

ϊ ΜΡί

Preparation of the LC / A-nocceptin-H fusion_N/TO -

<img file="MX337729B_D0095.tif" />

To create the LC construction - in laz-an-te- noeie e p4 inaespac¡ador-H_N (SEQ ID 13), the binding encoding pCR 4 vector (SEQ ID 7) is cleaved by the restriction enzymes of

BamHI + I went out. This broken vector then serves as the receptor vector for binding of the LC / A DNA and DNA (SEQ ID 1) broken with BamHI + Sali. The resulting plasmid DNA is then cleaved with the Xbal Pstl + restriction enzymes as the receptor vector for insertion and ligation of the H DNA._N/ A (SEQ ID 2) rotates with Pstl + Xbal. The final construction contains LC-linker-noccept-spacer-H_N ORF (SEQ ID 13) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ

ID 14.

Example 3 - Preparation of a nociceptin LC / AH fusion protein_N/ A (nociceptin is the N-terminus of the LC chain)

The LC / AH base_N/ A is constructed as described in Example 2 using the synthesized serotype A linker with the addition of a factor XA site for activation, arranged while BamHl-Sa / l-linker-protease-linker-PsflXbal- site. stop codon-H / ndIII (SEQ ID 8). The LC / A-HN / A base and the N-terminal synthesized nociceptin insert (SEQ ID 9) are cleaved with the restriction enzymes BamHI + Hindlll, gel purified and ligated to create a nociceptin-spacer-LC -binder-H_N. The ORF (SEQ ID 15) is then

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<img file="MX337729B_D0096.tif" />

cleaves using Aval + Xbal restriction enzymes for transfer into expression vectors so that expression results in a fusion protein of the sequence illustrated in SEQ ID

16.

Example 4 - Preparation of an LC / Cnocicepti na-H fusion protein_N/ C

Following the methods used in Examples 1 and 2, the LC / C (SEQ ID 5) and HN / C (SEQ ID 6) are created and inserted into the C serotype linker arranged as BamHl-Sall-linker protease-nociceptin site -Nhel-separator-Spel-Psfl-Xbai-stop codon-HindIII (SEQ ID 10). Final construction contains LC-linker-nociceptin-spacer-H_N ORF (SEQ ID 10) for expression as protein of the sequence illustrated in SEQ ID 18. Example 5 - Preparation of a LC / Cnociceptin-HN / C fusion protein with an activation sequence of serotype A

Following the methods used in Examples 1 and 2, the LC / C (SEQ ID 5) and HN / C (SEQ ID 6) are created and inserted into the C serotype linker arranged as BamHI-Sall-protease-nociceptin site -Nhel-separator-Spel-Psfl-Xbal-stop codon-HindIII (SEQ ID 7). Final construction contains LC-linker-nociceptin-spacer-H_N ORF (SEQ ID 19) for expression as protein of the sequence illustrated in SEQ ID 20. Example 6 - Preparation of an LC / A-met enkephalin-H fusion protein_N/TO

Due to the small size, five-amino acid, of the ligand

<img file="MX337729B_D0097.tif" />

met-enkephalin, the fusion of LC / A-met enkephalin-H_N/ A is created by site-directed mutagenesis [eg using Quickchange (Stratagene Inc.)] using the LC / A-nociceptin-H fusion_N/ A (SEQ ID 13) as a template. Oligonucleotides encoding the met-enkephalin peptide YGGFM are designed, ensuring that the standard use of the Escherichia Coli codon is maintained and that no additional restriction sites are incorporated, flanked by sequences complementary to the region of the LC / fusion linker Anociceptin-H_N/ A (SEQ ID 13) either side in the nociceptin section. The SDM product is checked by sequencing and that the final construct contained the enkephalin-LC-linker-met spacer-H_N ORF (SEQ ID 21) for expression as protein of the sequence illustrated in SEQ ID 22.

Example 7 - Preparation of an LC / Α-β endorphin-HNZA fusion protein

Following the methods used in Examples 1 and 2, LC / A (SEQ ID 1) and H are produced_N/ A (SEQ ID 2) and inserted into serotype A β-endorphin binder arranged as BamHISa / l-binding-protease site-β endorphin-Nhel-spacer-SpelPsfl-Xbal-stop codon-HIndIII (SEQ ID eleven). The final construct contains the LC-linker-endorphin-spacer-H_N ORF (SEQ ID 23) for expression as sequence protein illustrated in SEQ

ID 24.

Example 8 - Preparation of an LC / Anocic ptin variant -H fusion protein_N/ A vlp i in; - ;: i L.

Following the methods used in examples -1 - ys © '··· produces LC / A (SEQ ID 1) and Hn / A (SEQ ID binding of variant of nociceptin β serotype A arranged as BamHI-Sa / l-binding- protease-variant nocicpetin site Nhel-spacer-Spel-Psfl-Xbal-stop codon-HIndIII (SEQ ID

12). The final construct contains the LC-linker-variant of nociceptin-spacer-H_N ORF (SEQ ID 25) for expression as a sequence protein illustrated in SEQ ID 26.

Example 9 - Purification method for the LC / A-nociceptin-H fusion protein_N/TO

Thaw the falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E Coli BL21 cell paste. Dilute the thawed cell paste to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonify on ice 30 seconds on, 30 seconds off for 10 cycles with an energy of 22 microns ensuring that the sample remains cold. Centrifuge the lyzed cells at 18,000 rpm, 4 ° C for 30 minutes. Load the supernatant onto a chelating column loaded with NiSO₄ 0.1 M (20-30 ml column is sufficient) balanced with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazole, rinsing off the non-specific bound protein and eluting the fusion protein with 100 mM imidazole. Dialyze the eluted fusion protein with 5 L of 50mM HEPES pH 7.2, 200mM NaCl at 4 ° C overnight and measure the OD of the dialyzed fusion protein. Add 1 unit of factor XA per 100 pg of protein and

100

<img file="MX337729B_D0098.tif" />

<img file="MX337729B_D0099.tif" />

in a

I tv ί \ L melt and incubate at 25 ° C overnight. Load chelating column loaded with NiSO₄ 0.1 M (cKíumñT ^ T'TÜ '^^ JTTmr is sufficient) balanced with 50 mM HEPES pH 7.2, 200 mM NaCI. Wash the column at baseline with 50mM HEPES pH 7.2, 200mM NaCI. Using a 10 and 40 mM gradient of imidazole, rinse the non-specific bound protein and elute the fusion protein with 100 mM imidazole. Dialyze the eluted fusion protein against 5L of 50mM HEPES pH 7.2, 200mM NaCI at 4 ° C overnight and melt at approximately 2mg / ml, take aliquots of the sample and freeze at -2O ° C. Test the purified protein using OD, BCA, purity analysis and SNAP-25 determination.

Example 10 - Confirmation of TM agonist activity by measuring the release of substance P from neuronal cell cultures

materials

The substance Ρ EIA is obtained from R&amp;D Systems, UK.

Methods

The primary neuronal cultures of eDRG are established as previously described (Duggan et al., 2002). The release of substance P from the cultures is determined by the EIA, essentially as previously described (Duggan et al., 2002). The TM of interest is added to the neuronal cultures (established at least 2 weeks before treatment); Control cultures are performed in parallel by adding vehicle instead of TM. (100 mM KCI) basal release, together

101

<img file="MX337729B_D0100.tif" />

<img file="MX337729B_D0101.tif" />

with the total content of the cell lysate, of the substituta ¿[! &amp; ¿P jse for both control cultures and treated with TM. Substance P immunoreactivity is measured using Substance P enzyme immunoassay systems (Cayman Chemical Company, USA or R&amp;D Systems, UK) according to the manufacturer's Instructions.

The amount of the substance P released by the neuronal cells in the presence of the TM interest is compared to the release obtained in the presence and absence of 100 mM KCI. The stimulation of the release of substance P by the TM of Interest over the basic release, establishes that TM of Interest is an agonist ligand "; as defined in this description. If the stimulation of substance P release by TM of interest is desired it can be compared to a standard substance P release curve produced using the natural ORL-1 receptor ligand, nociceptin (Tocris). Example 11 - Confirmation of ORLj receptor activation by measuring forskolin stimulated production of cAMP

It is confirmed that the given TM is acting via the ORLt receptor is provided by the following test, in which the ability of TM to inhibit forskolin-stimulated cAMP production.

PHjadenine materials and [¹⁴C] CAMP is obtained from GE Healthcare

Methods

The test is conducted essentially as described

102

<img file="MX337729B_D0102.tif" />

previously by Meunier et al [isolation and structure of the endogenous opioid receptor type ORLi agonist. Nature 377: 532-535, 1995] in CHO-transfected cells inoculated in plastic plates with 24 wells.

Aggregated adenine [3H] (pCi 1.0) is added to the cells in 0.4 ml of culture medium. The cells remain at 37 ° C for 2 h to allow adenine to incorporate into intracellular ATP. After 2 hr, cells are washed once with incubation buffer containing: 130mM NaCl, 4.8mM KCI, 1.2mM KH₂PO₄1.3 mM CaCI₂1.2 mM MgSO₄, 10 mM glucose, 1 mg / ml bovine serum albumin and 25 mM HEPES pH 7.4, and replaced with bovine serum buffer containing forskolinine isobutylmethylxanthine (10 pM) and (μΜ 50) with or without TM of interest. After 10 minutes, the medium is aspirated and replaced with 0.5 ml, 0.2 M HCI. Approximately 1000 cpm of [¹⁴C] cAMP are added to each well and used as an internal standard. The contents of the wells are then transferred to the columns of 0.65g dry alumina powder. The columns are eluted with 4 ml of 5 mM HCI, 0.5 ml 0.1M ammonium acetate, then two additional milliliters of ammonium acetate. The final eluate is collected in scintillation flasks and counted for¹⁴C and tritium. The amounts collected are corrected for the recovery of [¹⁴C] cAMP. TM that are agonists at the ORLt receptor causes a reduction in the level of cAMP produced in response to forskolin.

Example 12 - Confirmation of the activation of the ORL1 receiver.

103

JL&gt;; · 'ί, «ν-, Using a functional analysis of nlac GTPyS. - .. MU ';'

Confirmation that given TM is acting via the ORL receiver! It is also provided by the following test, a functional analysis of GTPyS link.

Materials [³⁵S] GTPyS is obtained from GE Healthcare

Agglutinin-coated wheat germ (SPA) grains are sourced from GE Healthcare

Methods

This analysis is performed essentially as described by that of Traynor and Nahorski [modulation by guanosine-5 O- agonists (3 [³⁵S] thio) triphosphate that binds to the membranes of human SH-SY5Y neuroblastoma cells. Mol. Pharmacol.

47:848 - 854, 1995],

Cells are scraped from tissue culture plates in 20mM HEPES, 1mM ethylenediaminetetracetic acid, then centrifuged at 500 xg for 10-10 minutes.

The cells are resuspended again in this buffer and homogenized with a Polytron homogenizer.

The homogenate is centrifuged at 27,000 xg for 15 minutes, and the pellet is resuspended in buffer A, containing: 20mM HEPES, 10mM MgCI₂, 100 mM NaCI, pH 7.4. The suspension was recentrifuged at 20,000 xg and resuspended once more in buffer A. For binding analysis, the membranes (8-15pg protein) are incubated with [³⁵S] GTP S (50 PM), GDP (μΜ 10), with and

104

IΜ Ρ ί:

, :·· ; ·.

L. ·.

ί '?' Hee.

without the TM of interest, in a total volume of 1.0 ml, for 60 minutes at 25 ° C. Samples are filtered over counted filters and fiberglass as described for ligation analyzes. Example 13 - Preparation of an LC / Anociceptin-HN / A fusion protein (nociceptin is the N-terminus of the chain

H_n)

The linker-nociceptin-spacer insert is prepared as described in Example 2.

Preparation of the LC / A-nociceptin-H fusion_N/TO

To create the LC-binder-nociceptin-spacer-H construct_N (SEQ ID 13), the pCR 4 vector encoding the linker (SEQ ID 7) is cleaved with the restriction enzymes BamHI + Salí. This broken vector then serves as the container for insertion and the LC / A DNA link (SEQ ID 1) also rotates with BamHI + Sali. The resulting DNA plasmid is then cleaved with BamHI + Hindlll restriction enzymes and the LC / A-linker fragment inserted into a similarly broken vector containing a single multiple breeding site for BamHI, BamHI, Pstl, and Hindlll such as the vector of pMAL ((NEB)). H's DNA_N/ A (SEQ ID 2) is then cleaved with Pst + Hindlll restriction enzymes and inserted into the similarly broken construct of pMAL-LC / A-linker. The final construct contains the LC-linker-nociceptin-spacerH_n ORF (SEQ ID 13) for expression as protein of the sequence illustrated in SEQ ID 14.

<img file="MX337729B_D0103.tif" />

105

IMS

U ΜExample 14 - Preparation of a nociceptin-LC / AH fusion protein_N/ A (nociceptin is termiTíST ^ Tcle ^ Tá''chain

LC) To create the nociceptin-spacer-LC / AH construct_N/ A, a serotype A linker with the addition of a factor XA site for activation, arranged as a BamHI-Sallsite-linker-Psfl-Xibal-stop codon protease-H / ndIII (SEQ ID 8) is synthesized as described in Example 13. The pCR 4 vector encoding the linker is cleaved with the restriction enzymes of BamHI + Sali. This broken vector then serves as the container for insertion of the LC / A DNA and binding (SEQ ID 1) also rotated with BamHI + Sali. The resulting plasmid DNA is then cleaved with the Hindlll BamHI + restriction enzymes and the LC / A linker fragment inserted into a similarly broken vector containing the synthesized nociceptin insert of the N-terminal presentation (SEQ ID 9) . This construct is then cleaved with Aval + Hindlll and inserted into an expression vector such as the pMAL plasmid (NEB). H's DNA_N/ A (SEQ ID 2) is then cleaved with the Hindlll Pstl + restriction enzymes inserted into the similarly broken construct of pMAL-nociceptin-LC / A-linker. Final construction contains nociceptin-separator-LC / AH_N/ A ORF (SEQ ID 51) for expression as protein of the sequence illustrated in SEQ ID 52.

Example 15 - Purification of the preparation and of a family of the LC / A-nocic ptina-HN / A fusion protein with length

106

ΙΜΡΙ6

- w | ¡SS Γΐ U ¡Ω ΛίΙ · ./,! · .. aNO y, / .. Dt LA. χ&gt; l- DV 'IND ^ itliCAL variabl d I spacer

Using the same strategy employed in Example 2, a range of DNA linkers was prepared which encode nociceptin and the variable spacer content. Using one of a variety of reverse translation software tools [eg EditSeq best E. coli reverse translation (DNASTAR Inc), or Backtranslation tool v2.0 (Entelechon)] the DNA sequence encoding the linker-ligand region- spacer is determined. The restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-Sallen link te-potent site-nociceptin-N and l-sepa rator-S pei-PsflXbal-stop codon-HIndlII ( SEQ ID 53 to SEQ ID 57). It is important to ensure that the correct reading frame is maintained for the spacer, nociceptin, and restriction sequences and that the Xbal sequence is not preceded by bases, TC which would result in DAM methylation. The DNA sequence is examined as soon as the restriction sequence is incorporated and any additional sequences are manually removed from the remaining sequence ensuring that common use of the Escherichia Coli codon is maintained. Escheríchia Coli codon usage is determined by reference to computer programs such as Graphical Codon Usage Analyzer (Geneart), and the% GC content and rate of codon usage was determined by reference to published tables on codon usage (eg GenBank Issue 143, September 13, 2004). This sequence

107

<img file="MX337729B_D0104.tif" />

DNA optimized is then commercially synthesized (eg by Entelechon, Geneart or Sigma-Genosys) and is provided in vector pCR 4.

The dividers that were created include:

<td>Code</td><td>Binder protein sequence</td><td>SEQ ID of linker DNA</td>

<td>GS10</td><td>ALAGGGGSALVLQ</td><td> 53</td>

<td>GS15</td><td>ALAGGGGSGGGGSALVLQ</td><td> 54</td>

<td>GS25</td><td>ALAGGGGSGGGGSGGGGSGGGGSALVLQ</td><td> 55</td>

<td>GS30</td><td>ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ</td><td> 56</td>

<td>HX27</td><td>ALAAEÁAAKEAAAKÉAAAKAGGGGSALVLQ</td><td> 57</td>

Table 1

As an example, to create the LC / A-CPN (GS15) construction of the H fusion_N/ A (SEQ ID 58), the pCR 4 vector encoding the linker (SEQ ID 54) is cleaved with the Sali BamHI + restriction enzymes. This broken vector then serves as the receptor vector for insertion and the LC / A DNA link (SEQ ID 1) also broken with the BamHI + Sali. The resulting plasmid DNA is then cleaved with the restriction enzymes BamHI + Hindlll and the LC / A-linker fragment inserted into a similarly broken vector that contained a single multiple site of reproduction for Salí, Pstl and Hindi such as the vector pMAL ((NEB)). H's DNA_N/ A (SEQ ID 2) is then cleaved with the Hindlll Pstl + restriction enzymes inserted into the similarly broken pMAL-LC / A-linker construct. The final construction contains the LC / A-CPN (GSI 5) - H_N/ A ORF (SEQ ID

108

<img file="MX337729B_D0105.tif" />

) for expression as protein of the sequence illustrated in

SEQ ID 59.

As another example, create the LC / A-CPN (GS25) - H fusion construct_N/ A (SEQ ID 60), the pCR 4 vector encoding the linker (SEQ ID 55) is cleaved by restriction enzymes

BamHI + I went out. This cleaved vector then serves as the receptor vector for insertion and the BamHI + cleaved LC / A DNA link (SEQ ID 1) exited. The resulting plasmid DNA is then cleaved with the enzymes and the LC / A-linker fragment inserted into a similarly broken vector containing a unique multiple breeding site for BamHI, Sali, Pstl, and Hindlll such as the pMAL vector ((NEB)). H's DNA_N/ A (SEQ ID 2) then breaks with the Hindlll restriction enzymes Pstl + and inserts into the similarly broken construct of the pMAL-LC / A- linker,

The final construction contains the LC / A-CPN (GS25) - H_N/ A ORF (SEQ ID 60) for expression as protein of the sequence illustrated in SEQ ID 61. The fusion variants consisting of GS10, GS30 and HX27 of LC / A-CPN-HN / A are created similarly. Using the purification methodology described in Example 9, the fusion protein is purified with the E. coli cell paste. Figure 9 illustrates the purified product obtained in the case of LC / A-CPN (GS10) - H_n/ A, LCVA-CPN (GSI 5) - Hn / A, LC / A-CPN (GS25) - H_N/ A, LC / A-CPN (GS30) - H_N/ A and LC / A-CPN (HX27) - H_N/TO.

Example 16 - Determination of the in vitro efficacy of an LC / A-nocic ptina-HN / A fusion

109

<img file="MX337729B_D0106.tif" />

The fusion protein prepared according to Examples 2 and 9 was determined in the eDRG cellular neuronal model.

Assays for inhibition of the release and cleavage of SNAP-25 substance P have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277, 34846-34852).

Briefly, dorsal root ganglia neurons are harvested from 15-day dissociated and fetal cells from Sprague-Dawley rats inoculated on 24 well plates coated with Matrigel at a density of 1 x 106 cells / well. One day after seeding the cells are treated with 10 µΜ cytosine β-Darabinofuranoside for 48 hours. Cells are maintained in Dulbecco's minimal essential medium supplemented with 5% heat-inactivated fetal calf serum, 5 mM L-glutamine, 0.6% D-glucose, 2%, B27 supplement, and 100 ng / ml factor growth nerve of the 2.5S mouse. Cultures are kept for 2 weeks at 37 ° C in 95% air / 5% CO₂ before adding test materials.

Release of Substance P from eDRG is determined by enzyme-linked immunosorbent analysis. Briefly, the eDRG cells are washed twice with the low potassium balanced salt solution (BSS: 5mM KCI, 137mM NaCl, 1.2mM MgCI2, 5mM glucose, 0.44mM KH₂PO₄, 20 mM HEPES, pH 7.4, 2 mM CaCI₂). Basic samples are obtained by incubating each well for 5 minutes, with 1 ml of BSS with low potassium. After removal of this buffer, cells are stimulated to release by the

110 AMPI

INSTITUTO MEXICANO DE LA t'KC'KEDAD incubation with 1 ml of the high potassium buffer^iL'fBiSS cbrrro' above with modification to include 100mM KCf isotonrcamenté 'balanced with NaCI) for 5 minutes. All samples are removed to tubes on ice prior to analysis of Substance P. Total cell shades are prepared by the addition of 250 µl of 2M acetic acid / 0.1% trifluoroacetic acid to break the cells, centrifugal evaporation, and resuspension in 500 pl of analysis buffer. Diluted samples are determined for substance P content. Substance P immunoreactivity is measured using Substance P enzyme immunoassay kits (Cayman Chemical Company or R&amp;D Systems) according to instructions. Substance P is expressed in pg / ml relative to a standard substance P curve performed in parallel.

Western blot and SDS-PAGE analyzes were performed using standard protocols (Novex). The SNAP-25 proteins were redissolved in 12% Tris / glycine polyacrylamide gel (Novex) and subsequently transferred to the nitrocellulose membrane. The membranes were probed with a monoclonal antibody (SM 1-81) that recognizes SNAP-25 broken and intact. The specific binding was visualized using peroxidase conjugated secondary antibodies and a chemiluminescent detection system. SNAP-25 rupture was quantified by exploring densitometry ((Molecular Dynamics Personal SI, ImageQuant data analysis software). The percentage of SNAP-25 rupture was calculated according to the formula: (SNAP rota- 25 / (SNAP-25 lll

<img file="MX337729B_D0107.tif" />

rotate and contact)) x100. ______

Following eDRG neurons following exposure to an LC / A-nociceptin-H fusion_N/ A (called CPN-A), both inhibition of cleavage and release of substance P from SNAP-25 are observed (Figure 10). After 24h exposure to fusion, 50% maximum release of SNAP-25 is achieved by a fusion concentration of 6.3 ± 2.5 nM.

The effect of fusion is also determined at defined time points after 16 hr exposure of eDRG to CPN-A. Figure 11 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still being observed at 28 days post-exposure.

Example 17 - Determination of the in vitro efficacy of a variable fusion of LC / A-nociceptin H_N/TO

The fusion protein prepared according to Examples 8 and 9 was determined in the cellular neural mode of eDRG using the method * described in Example 16.

After exposure of eDRG neurons to a variant-H fusion_NWith LC / A-nociceptin (called VNCP-A), both inhibition of cleavage and release of substance P from SNAP25 are observed. After 24 h exposure to the fusion, 50% of the maximum SNAP-25 release is achieved by a fusion concentration of 1.4 ± 0.4 nM (Figure 12).

The effect of the fusion is also determined at the defined points of time after giving a 16 h exposure of eDRG to

112

<img file="MX337729B_D0108.tif" />

CPN-A. Figure 13 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still observed at 24 days post-exposure.

The binding capacity of the CPNv-A fusion protein is also determined with respect to the CPN-A fusion. Figure 14 illustrates the results of a competition experiment to determine binding efficiency at the ORL-1 receptor. CPNv-A is shown to displace [3H] -nociceptin, thereby confirming that access to the receptor is possible with the ligand in the central format of the presentation.

Example 18 - Preparation of a nociceptin-H LC / Avariant fusion protein_N/ A that is activated by treatment with Enterokinase

According to the methods used in Examples 1 and 2, LC / A (SEQ ID 1) and H_N/ A (SEQ ID 2) are created and inserted into the BamHI-Sallenlazante-Enterokinase site of protease-variant of nociceptin Nhel-separator-Spel-Psfl-Xbal-stop codon-HindIII (SEQ ID 62).

The final construct contains the LC-linker-variant nociceptin-spacer-H sequences._N ORF of (SEQ ID 63) for expression as protein of the sequence illustrated in SEQ ID 64. The fusion protein is called CPNv (Ek). Figure 15 illustrates the purification of E. coli CPNv (Ek) -A according to the methods used in Example 9 but using Enterokinase for activation at 0.00064 pg per 100 pg of the fusion protein.

Example 19 - Determination of the in vitro efficacy of a fuion

113

<img file="MX337729B_D0109.tif" />

LC / A-variant of nocic ptina H_N/ What has been activated by the treatment with enterokinase

The CPNv (Ek) - prepared in Example 18 is obtained in a purified form and applied to the eDRG cell model to determine SNAP-25 release (using the methodology of Example 16). Figure 16 illustrates the release of SNAP-25 after 24 h of exposure of eDRG to CPNv (Ek) -A. The efficacy of the release is observed to be similar to that achieved with the material broken with factor XA, as recorded in Example 17.

Example 20 - Preparation of a nociceptin variant LC / C fusion protein with a factor XA activation linker derived from serotype A

According to the methods used in Example 4, LC / C (SEQ ID 5) and H_N/ C (SEQ ID 6) are created and inserted into the BamHI-Sall-linker-nociceptin variant Nhel-spacer-SpelPsfl-Xbal-stop codon-HindIII (SEQ ID 65). The final construct contains the LC-linker-variant nociceptin spacer-H sequences_N ORF of (SEQ ID 66) for expression as protein of the sequence illustrated in SEQ ID 67. The fusion protein is called Cpnv.-C (act. A). Figure 17 illustrates the purification of Cpnv-C (act A) from E. Coli according to the methods used in Example 9

Example 21 - Determination of the in vitro efficacy of an LC / C fusion protein - variant of nociceptin HN / C

According to the methods used in Example 9, CPNv-C

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(act. A) prepared in Example 20 is obtained in Ciña fó'rmá purified and applied to the model of the eDRG cell to detérmínar'Tá release of SNAP-25 (using the methodology of Example 16). After 24 h exposure to the fusion, 50% maximum syntaxin release is achieved by a fusion concentration of 3.1 ± 2.0 nM. Figure 18 illustrates the release of syntaxin after 24 h of exposure of eDRG to CPNv-C (act. A).

Example 22 - Determination of the in vivo efficacy of an LC / A-nociceptin-HN / A fusion

The capacity of an LC / A-nociceptin-H fusion_N/ A (NPC / A) to inhibit acute mechanical allodynia induced by capsicin is evaluated after subcutaneous transplantation injection into the hind paw of the rat. Test animals are evaluated for paw removal frequency (PWF%) in response to 10 g of Von Frey filament stimulus series (10 stimuli x 3 trials) before study recruitment, after treatment subcutaneous with CPN / A but before capsaicin, and after the challenge with post-injection CPN / A capsian (average responses in 15 'and 30'). Capsicin challenge met

<td>by injection</td><td>from 10 pL of</td><td colspan="2">0.3% solutions.</td><td>Dilutions of</td><td>the</td>

<td colspan="2">sample prepare in 0</td><td>.5% BSA / saline.</td><td>The</td><td>figure 19 illustrates</td><td>the</td>

<td>investment of</td><td>allodynia</td><td>mechanics that</td><td>is</td><td>reached for</td><td>the</td>

<td>treatment</td><td>prior to</td><td>animals</td><td colspan="2">with a range</td><td>of</td>

LC / A-noclceptin-H fusion concentrations_N/TO.

The capacity of an LC / A-nociceptin fusion is evaluated115

<img file="MX337729B_D0111.tif" />

H_n/ A (CPN / A) to inhibit streptozotocin-induced (tactile) allodynia (STZ) - in rats. STZ-induced mechanical allodynia in rats is achieved by injection of streptozotocin (ip or iv) which results in the destruction of pancreatic β cells leading to loss of insulin production, with concomitant metabolic stress (hyperlipidemia and hyperglycemia). As such, STZ induces type I diabetes. In addition, STZ treatment leads to the progressive development of neuropathy, which serves as a model of chronic pain with allodynia from hyperalgia and may reflect samples seen in diabetic humans (peripheral diabetic neuropathy).

Male Sprague-Dawley rats (250-300 g) are treated with 65 mg / kg STZ in citrate (IV) buffer and blood glucose and lipids are measured weekly to define model adequacy. The paw withdrawal threshold (PWT) is measured in response to a series of Von Frey filament stimuli over a period of time. Allodynia is said to have been established when the PWT on two consecutive test dates (separated by 1 week) measures below 6 g on the scale. At this point, rats are randomly selected into a saline group (negative efficacy control), the gabapentin group (positive efficacy control), or a test group (NPC / A). Test materials (20-25 pl) are injected subcutaneously while a single injection (except gabapentin) and PWT is measured at 1 day post-treatment periodically thereafter for a period of two weeks.

116 ml / kg injection volume); be before the start of the test; of

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Gabapentin (30 mg / kg ip ($. Injected daily, 2 hours.

PWT. Figure 20 illustrates the inversion of qlodynia achieved by pretreatment of the animals with 750 ng of CPN / A. Data were obtained over a two-week period after a single injection of CPN / A.

Example 23 - Determination of the in vivo efficacy of an LC / A-variant nociceptin H fusion_n/TO

The ability of an LC / A-variant nociceptin HN / A (CPNv / A) fusion to inhibit capsicin-induced mechanical allodynia is evaluated after subcutaneous intraplantar injection in the hind paw of the rat. Test animals are evaluated for paw withdrawal frequency (PWF%) in response to a series of 10 g Von Frey filament stimuli (10 stimuli x 3 trials) prior to study recruitment (pre- treatment); after subcutaneous intraplantar treatment with CPNv / A but before capsaicin (Pre-CAP); and after the challenge with capsicin and post-injection CPNv / A (average responses in 15 'and 30'; CAP). The challenge of capsicin is achieved by injection of 10 pl of 0.3% solutions. Sample dilutions are prepared in 0.5% BSA / saline. Figure 21 illustrates the reversal of allodynia qqe is achieved by pretreating animals with a range of LC / A-variant fusion concentrations, "je nociceptin-HN / A with respect to the inversion achieved with the addition of LC / A-noc¡cept¡na fusion- $_N/TO. These data are

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expressed as a normalized differential of the frequency of paw withdrawal, in which the difference between the maximum response (postcapsicin) and the response of the baseline (precapsicin) is expressed as a percentage. With this analysis, it can be seen that CPNv / A is more potent than CPN / A since a lower dose of CPNv / A is required to achieve analgesic effect similar to that compared to CPN / A.

Example 24 - Preparation of an LC / Aleu enkephalin-HN / A fusion protein

Due to the small size, five amino acids, of the leu-enkephalin ligand the LC / A-leu enkephalin-H fusion_N/ A is created by site-directed mutagenesis [eg using Quickchange (Stratagene Inc.)] using the LC / A-nociceptin-H fusion_N/ A (SEQ ID 13) as a template. Oligonucleotides encoding the YGGFL leu-enkephalin peptide are designed, ensuring that the standard use of the E Coli codon is maintained and that no additional restriction sites are incorporated, flanked by sequences complementary to the region of the linker of the LC / Anociceptin-H fusion_N/ A (SEQ ID 13) either side in the nociceptin section. The SDM product is verified by sequencing and the final construct containing the enkephalin-LC-linker-leu Spacer-H_N ORF (SEQ ID 68) for expression as a protein of the sequence illustrated in SEQ ID 69. The fusion protein is called CPLE-A. Figure 22 illustrates purification of E. Coli CPLE-A according to the methods used in Example 9.

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<img file="MX337729B_D0113.tif" />

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Example 25 - Purification of the expression and protein of the LC / A-beta-endorphin-H fusion_N/TO

According to the methods used in Example 9, and with the LC / A-beta-endorphin-H fusion protein_N/ A (called CPBEA) created in Example 7, CPBE-A is purified from E. Coli. Figure 23 illustrates the purified protein as analyzed by SDSPAGE.

Example 26 - Preparation of a mutant LC / Anociceptin-H fusion protein_N/TO

Due to the only amino acid modification necessary to mutate the nociceptin sequence at position 1 from a Phe to a Tyr, the LC / A-mutant nociceptin-HN / A fusion is created by site-directed mutagenesis [eg using Quickchange (Stratagene Inc.)] using the LC / A-nociceptin-H fusion_N/ A (SEQ ID 13) as a template. Oligonucleotides are coding tyrosine designed at position 1 of the nociceptin sequence, ensuring that the standard use of the E Coli codon is upheld and that no additional restriction sites are incorporated, flanked by sequences complementary to the linker strand of LC / A-nociceptin-H fusion_N/ A (SEQ ID

13) anywhere in the nociceptin section. The SDM product is checked by sequencing and the final construct containing the ORF (SEQ ID 70) fusion of LC / A-nociceptin mutant spacer H_N/ A for expression as protein of the sequence illustrated in SEQ ID 71. The fusion protein is called CPOP-A. The

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<img file="MX337729B_D0115.tif" />

Figure 24 illustrates purification of CPOP-A from E. Coli according to the methods used in Example 9.

Example 27 - Determination of the preparation and a variable protein of the mutant LC / A-nociceptin-H fusion_N/TO

Due to the single amino acid modification necessary to transform the nociceptin sequence at position 1 from a Phe to a Tyr, the LC / A-variable nociceptin mutant H fusion_N/ A is created by site-directed mutagenesis [eg using Qulckchange (Stratagene Inc.)] using the LC / Anociceptin variant-H fusion_N/ A (SEQ ID 25) as a template. Oligonucleotides are coding tyrosine designed at position 1 of the nociceptin sequence, ensuring that the standard use of the E Coli codon is maintained and that no additional restriction sites are incorporated, flanked by the complementary sequences to the linker region of LC / Avariante fusion of noc¡cept¡na-H_N/ A (SEQ ID 25) anywhere in the nociceptin section. The SDM product is checked by sequencing and the final construct containing the ORF (SEQ ID 72) fusion of LC / A-mutant-spacer-H-nociceptin_N/ A for protein expression of the sequence illustrated in SEQ ID 73. The fusion protein is called CPOPv-A. Figure 25 illustrates purification of CPOPv-A from E Coli according to the methods used in Example 9.

Using the methodology described in Example 16, CPOPv-A is determined so that its capacity breaks SNAP-25 in the model

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<img file="MX337729B_D0116.tif" />

eDRG cell phone. Figure 26 illustrates that CPOPv-A can break SNAP-25 in the eDRG model, achieving 50% maximum release of SNAP-25 after exposure of cells to fusion of approximately 5.9 nM for 24H.

Example 28 - Preparation of an IgA protease-variant nociceptin-H fusion protein_N/TO

The amino acid sequence of the IgA protease was obtained from freely available sources in databases such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease IgA gene is available in the literature (Pohlner et al., Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature, 1987, 325 (6103), 458- 62). Using Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the modified IgA protease for E Coli expression was determined. A BamHI recognition sequence was incorporated at the 5 'end and a codon encoding an amino acid and cysteine ​​Sali recognition sequence was incorporated at the 3'end of IgA DNA. The DNA sequence was screened using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during subsequent translation. Any cleavage sequences that are common to those required for cloning were manually removed from the proposed coding sequence which ensures that common use of the E. coli codon is maintained. The use of the E. Coli codon was

121

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IN'DU.-T.UAL determined by Graphical Analyzer of Codon Use (Geneart), and the% GC contained and the proportion of codon use was determined by reference to the published tables of codon use. This optimized DNA sequence (SEQ ID 74) containing the IgA open reading frame (ORF) is then commercially synthesized. IgA (SEQ ID 74) is inserted into the LC-linker variant of nociceptin-spacer-H_N ORF (SEQ ID 25) using the restriction enzymes I came out of BamHI and to replace the LC with the IgA protease DNA. Final Construction Contains IgA Linker-Nociceptin Variant-Spacer-H_N ORF (SEQ ID 75) for expression as protein of the sequence illustrated in SEQ

ID 76.

Example 29 - Determination of the preparation and of a protein of the endopeptidase fusion directed to nociceptin with a detachable label of the purification of histidine.

DNA encoding a removable his factor XA (hίεβ) tag was prepared, although it is clear that alternative protease site such as alternative Enterokinase purification tags such as longer histidine tags is also possible. Using one of a variety of reverse translation software tools [eg EditSeq best E. coli reverse translation (DNASTAR Inc), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the detachable region of the his-tag of factor XA is determined. The restriction sites are then incorporated into the DNA sequence and

122

<img file="MX337729B_D0118.tif" />

<img file="MX337729B_D0119.tif" />

can fix as Nhel-binding-Spel-Pstl-H_N/ A-Xbal- stop codon-LElEGRSGHHHHHH-Hindll (SEQ ID 77). LT ^ DNA sequence is examined for the built-in restriction sequence and any additional sequences are manually removed from the remaining sequence ensuring that common use of the E. coli codon is maintained. The use of the E. codon Coli is determined by reference to computer programs such as the Graphical Codon Usage Analyzer (Geneart), and the% GC of the content and codon usage was determined by reference to the published Codon Usage Tables (eg issue 143 , September 13, 2004 from GenBank). This optimized DNA sequence is then commercially synthesized (eg by Entelechon, Geneart or Sigma-Genosys) and is provided in vector pCR 4. To create CPNv-A-FXa-HT (SEQ ID 78, his-tag detachable construct) the pCR 4 vector encoding the detachable-his tag is cleaved with Nhel and Hindlll. The Hindlll fragment is then inserted into the LC / A-CPNv-HN / A vector (SEQ ID 25) which has also been disrupted by Nhel and Hindlll. The final construct contains the LC / A-linker-variant nociceptinase spacer-HN-FXa-Histag-HindlII ORF (SEQ ID 78) sequences for protein expression of the sequence illustrated in SEQ ID 79. Figure 27 illustrates the purification of E Coli CPNv-A-FXa-HT according to the methods used in Example 9.

Example 30 - Preparation of ndopeptide a fusion protein labeled with an I u- nc phalin containing a

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JND'JEJ »domain of tran d location derived from toxin of ia ^ clífferia '

The DNA sequence is designed by subsequent translation of the diphtheria toxin translocation domain amino acid sequence (obtained from freely available database sources such as GenBank (accession number 1XDTT) using one of a variety of tools post translation software [eg EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. The restriction sites are then incorporated into the sequence and DNA can be arranged as a Nhel -Spel-Psfl-diphtheria translocation domain-Xbal-stop codon Hindlll (SEQ ID 80). The Pstl / Xbal recognition sequences are incorporated at the 5 'and 3' ends of the translocation domain respectively of the sequence that maintains the correct reading frame. The DNA sequence is examined (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during subsequent translation. Any break sequences that are found are common to those required by the breeding system to be manually removed from the proposed coding sequence ensuring that the use of the common E Coli codon is maintained. E Coli codon usage is determined by reference to computer programs such as graphical codon usage analyzer (Geneart), and% GC of codon content and usage was determined by reference to published usage tables of the codon (for

124

<img file="MX337729B_D0120.tif" />

example edition 143, September 13, 2004 of GenBank). This optimized DNA sequence containing the diphtheria translocation domain is then synthesized commercially as NHel-linker-Spel-Psfldomain-Xbal-stop codon-HindIII diphtheria shift (eg by Entelechon, Geneart or Sigma Genosys ) and provided in vector pCR 4 (Invitrogen). The pCR 4 vector encoding the diphtheria translocation domain is cleaved with Nhel and XbaL. The Nhel-Xbal fragment is then inserted into the LC / A-CPLE-H vector_N/ A (SEQ ID 68) which has also been broken by Nhel and Xbal. The final construct contains the ORF sequences of the LC / A-leu-enkephalin-spacer-diphtheria translocation domain (SEQ ID 81) for protein expression of the sequence illustrated in SEQ

ID 82.

Example 31 - Preparation of Endopeptidase Fusion Protein Labeled with a Nociceptin Variant Containing a Translocation Domain Derived from Tetanus Toxin

The DNA sequence is designed by subsequent translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as GenBank (accession number X04436) using one of a variety of identification tools). reverse translation software [eg EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)].

125

<img file="MX337729B_D0121.tif" />

BamHI / Sall recognition are incorporated at the 5 'and 3' ends respectively of the sequence that maintains the correct reading frame (SEQ ID 83). The DNA sequence is examined (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during subsequent translation. Any break sequences found to be common to those required by the cloning system are maintained and manually removed from the proposed coding sequence that ensures common use of the E Coli codon. Escherichia Coli codon usage is determined by reference to computer programs such as graphical codon usage analyzer (Geneart), and and% GC of codon content and usage was determined by reference to published usage charts. codon (eg GenBank issue 143, September 13, 2004). This optimized DNA sequence containing the tetanus LC toxin open reading frame (ORF) is then commercially synthesized (eg by Entelechon, Geneart or Sigma-Genosys) and provided in vector pCR 4 (invitrogen). The pCR 4 vector encoding TeNT LC is cleaved with BamHI and Sali. The BamHI-Salí fragment is then inserted into the LC / ACPNv-H vector_n/ A (SEQ ID 25) which has also been broken with BamHI and Salí.

The final construct contains the TeNT LC- variant nociceptin spacer-H sequences_N (SEQ ID 84) for expression as protein of the sequence illustrated in SEQ ID 85.

Example 32 - Pr ation of a protein from the LC / C126 fuion

<img file="MX337729B_D0122.tif" />

<img file="MX337729B_D0123.tif" />

native ti d I variant of nocic ptina-H_N/ C with a nlazante serotype C that is susceptible to breakdown with XA factors

According to the methods used in Example 4, the LC / C (SEQ ID 5) and HN / C (SEQ ID 6) are created and inserted into the nociceptin variant linker of the C serotype arranged as SamHISall-in lazant- Nociceptin-Nhel-Separator-Spel-PsflXibal-Stop Codon-HindIII variant (SEQ ID 86). The final construct contains the LC-linker-variant nociceptin spacer-H ORF sequences_N of (SEQ ID 87) for expression as protein of the sequence illustrated in SEQ ID 88. The fusion protein is called CPNv-C (act. C).

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<img file="MX337729B_D0124.tif" />

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1.

A