Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics

Definitions

• the present invention is directed to the use of Iml and Mil conotoxin peptides, and derivatives thereof, as cardiovascular agents including, but not limited to, heart rate regulating agents, blood pressure regulating agents and anti-arrhythmia agents.
• nAChRs nicotinic acetylcholine receptors
• the -conotoxins belong to a family of structurally related peptides found in the venom of marine snails of the genus Conus. These peptides target nAChRs and are promising ligands for investigating different subtypes of nAChRs. Two of these toxins, ⁇ -conotoxin Mil (from Conus magus) and ⁇ -conotoxin Iml (from Conus imperialis), were recently described, and they discriminate among different combinations of nAChR subunits expressed in Xenopus oocytes injected with RNA cloned from rat brain (Johnson et al., 1995, Cartier et al., 1996). The structures of these peptides are set forth below.
• B neurones are innervated by axons in the connective above the 7th ganglion, whereas C neurones are innervated by axons in the rami of 7th and 8th ganglia (see review by Skok, 1973; also Dodd & Horn, 1983a).
• B and C neurones express both nicotinic and muscarinic ACh receptors. Nicotinic receptors mediate the fast epsp in both classes of neurones; however, the muscarinic receptors in B neurones mediate a slow epsp, and those in C neurones mediate a slow ipsp (Dodd & Horn, 1983b).
• nAChRs with different channel kinetics
• the heart does not require innervation in order to beat.
• the heart does have a nervous supply.
• Cardiac muscle like smooth muscle and unlike skeletal muscle, has a dual innervation from the autonomic nervous system.
• Parasympathetic nerves from the tenth cranial nerve (vagus) and sympathetic nerves from a ganglion of the paravertebral trunk both reach the heart. Stimulation of the vagus nerve slows the rate of heart beat, whereas stimulation of the sympathetic nerves to the heart speeds it.
• the stroke rate at any given time then, depends upon the ratio of nerve impulse frequencies along these two routes to the heart. In addition, the stroke rate also affects the blood pressure.
• the tenth paravertebral sympathetic ganglion has two types of neurones ⁇ B cells and C cells.
• the nicotinic synapses on B and C cells do have different safety margins, and these safety margins are differentially use-dependent.
• C cells have identical sensitivities to d-tubocurarine.
• the intrinsic safety margins of the nicotinic synapses of B and C neurones are not static but are dynamically influenced by pre- and post- synaptic modulation.
• Cardiovascular agents have been developed to treat patients having abnormal heart rate or abnormal blood pressure. It is desired to develop additional cardiovascular agents which are more effective than prior agents and which can discretely affect the heart rate or blood pressure without affecting other muscles.
• the present invention is directed to the use of Iml and Mil conotoxin peptides, and derivatives thereof, as cardiovascular agents including, but not limited to, heart rate regulating agents, blood pressure regulating agents and anti-arrhythmia agents.
• Figure 1 shows a sketch of the recording chamber.
• Figure 2 shows that nicotinic antagonists can differentially block synaptic transmission of B and C neurones in the 10th sympathetic ganglion of frog.
• the figure shows the peak amplitudes of B waves (closed circles) and C waves (open circles) plotted against time.
• ⁇ -Conotoxin Iml (5 ⁇ M) almost completely blocked B waves (by 95%), but blocked C waves only slightly (by 21%).
• ⁇ -Conotoxin Ml blocked B and C waves by only 26% and 25%, respectively
• d- Tubocurarine (dTC, 10 ⁇ M) almost completely blocked B waves (by 95%), but blocked C waves only slightly (by 26%).
• ⁇ -Conotoxin Mil (5 ⁇ M) blocked B waves by 37%, but blocked C waves nearly completely (by > 94%).
• Dihydro- ⁇ -erythroidine (D ⁇ E, 5 ⁇ M) blocked both B and C waves almost completely (by 95% and > 94%, respectively).
• Figure 3 shows traces of responses just before (thin solid curve), during (bold curve) and after (dashed curve) exposure to cholinergic antagonists. The responses were obtained during the experiment illustrated in Figure 2.
• the horizontal line represents the baseline (zero ⁇ V).
• the stimulus to the preganglionic nerve was presented 5 ms from the start of each trace (see arrow in
• Figure 3A note stimulus artifact at this location in all panels). In control responses (thin solid curve in each panel) the first deflection after stimulus artifact is the fast B wave. It is followed by the smaller slow B wave, then the double-humped C wave.
• Figure 3 A shows that ⁇ -conotoxin Iml is more effective in blocking B than C waves. Bold response was obtained 18 minutes after addition of 5 ⁇ M ⁇ -conotoxin Iml, and dashed response after 45 minutes of washing.
• Figure 3B shows that ⁇ -conotoxin Ml is not very effective in blocking either B or C waves. Bold response was obtained 55 minutes after addition of 5 ⁇ M ⁇ -conotoxin Ml, and dashed response after 22 minutes of washing.
• Figure 3C shows that d-tubocurarine is more effective in blocking B than C waves.
• Bold response was obtained 16 minutes after addition of 10 ⁇ M d-tubocurarine, and dashed response after 30 minutes of washing.
• Figure 3D shows that ⁇ -conotoxin Mil is much more effected in blocking
• the present invention is directed to the use of Iml and Mil conotoxin peptides, and derivatives thereof, as cardiovascular agents including, but not limited to, heart regulating agents, blood pressure regulating agents and anti-arrhythmia agents.
• Iml and Mil have the following formulas:
• the C-terminus is preferably amidated.
• the C-terminus is preferably amidated.
• the proline residues can be substituted with hydroxy-proline without affecting the biological activity of the peptides.
• Asn 5 can be substituted with His or Tyr and/or His 12 can be substituted with Asn or Tyr.
• Each peptide contains two disulfide bonds between the first and third and the second and fourth cysteine residues.
• SCLC tumors is described in U.S. Patent No. 5,595,972, incorporated herein by reference.
• ⁇ -conotoxin Mil and ⁇ -conotoxin Iml two new acetylcholine receptor antagonists, ⁇ -conotoxin Mil and ⁇ -conotoxin Iml, on nicotinic synaptic transmission in the 10th paravertebral sympathetic ganglion of the leopard frog (Rana pipiens) were examined.
• the preganglionic nerve was electrically stimulated (at low frequency, ⁇ 1 min " ', to avoid use-dependent changes) while compound action potentials of B and C neurones (B and C waves, respectively) were monitored extracellularly from the postganglionic nerve.
• ⁇ -conotoxins Mil and Iml at low micromolar concentrations, reversibly blocked both B and
• nicotinic antagonists can differentially block synaptic transmission of B versus C neurones with opposite selectivities strongly suggests that these neurones possess distinct nicotinic receptors.
• compositions containing a compound of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remingto 's Pharmaceutical Sciences. 18th Ed. (1990, Mack Publishing Co., Easton, PA). Typically, an antagonistic amount of the active ingredient will be admixed with a pharmaceutically acceptable carrier.
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral or parenteral.
• the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
• any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
• tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
• the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
• suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
• the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
• the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
• the active agents can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
• a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
• Suitable delivery systems are described in U.S. Patent No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503,
• the DNA sequence can be isolated from cDNA libraries using degenerate probes based on the sequences of the active agents herein or amplified from these libraries using appropriate degenerate primers. Alternatively, suitable DNA sequences can be prepared synthetically for each of the disclosed active agents on the basis of the disclosed sequences and the known genetic code.
• the active agents of the present invention are administered in an amount sufficient to generate the desired cardiovascular effect.
• the dosage range at which these agents exhibit this effect can vary widely, depending upon the severity of the patient's defect, the patient, the route of administration and the presence of other underlying disease states within the patient.
• the active agents exhibit their therapeutic effect at a dosage range from about 0.05 mg/kg to about 250 mg/kg, and preferably from about 0.1 mg/kg to about 100 mg/kg of the active ingredient.
• a suitable dose can be administered in multiple sub-doses per day.
• a dose or sub-dose may contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form.
• a more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form.
• Lumbar paravertebral ganglia 7 through 10 and the adjoining 10th spinal nerve were isolated from adult frogs (Rana pipiens) of both sexes.
• the recording chamber was fabricated from Sylgard, and consisted of a trough about 20 mm long, 3 mm wide and 2 mm deep, which could be divided into four compartments A-D by three partitions 1-3. Three vertical, transverse slits were cut into the wall and floor of the trough with a 10 mm- wide razor blade.
• the trough was partitioned by inserting into each slot an approximately 8 mm wide x 3 mm high x 0.1 mm thick sheet of Mylar, with a V-shaped notch in the top edge.
• each sheet penetrated the floor of the trough so the vertex of the notch rested slightly above the floor.
• the sympathetic chain was pinned to the bottom of the trough.
• ganglia are represented by hatched ovals and the 10th spinal nerve by a broad stippled line.
• the 7th and 8th ganglia were located in compartment A, and the 9th and 10th ganglia in compartment B.
• Segments of the 10th nerve (stippled line) containing post-ganglionic axons of neurones in the 10th ganglion traversed partitions 2 and 3.
• the middle segment of the 10th spinal nerve was located in compartment C, and the caudal stump of the
• compartment D 10th spinal nerve was located in compartment D.
• Partition 2 effectively reduced the volume of the compartment containing the 10th ganglion and thereby reduced the amount of toxin required; moreover, it helped to minimize stimulus artifacts.
• compartment B -30 ⁇ l volume
• the chain of ganglia and the 10th spinal nerve were draped over the notched partitions and pinned to the floor of the trough.
• Postganglionic compound action potentials of B and C neurones in the 10th ganglion were recorded from the 10th spinal nerve with Pt wire electrodes placed on either side of partition 3 and connected to a high-input impedance differential A/C preamplifier (either P-15, Grass Instruments or DAM-50, WP Instruments, with 1 Hz low and 1 kHz high frequency filters).
• the recording electrode in compartment D led to the positive input of the preamplifier, while that in C led to the negative input.
• a Pt wire ground electrode was located in compartment B.
• Stimuli (1 ms rectangular voltage pulses) were provided by a stimulator (S-88, Grass Instruments) through a stimulus isolation unit.
• the preparation was bathed in frog Ringer's solution consisting of 111 mM NaCl, 2 mM
• Synthetic ⁇ -conotoxins (Mclntosh et al., 1994; Cartier et al., 1996) were used, d- Tubocurarine was from Sigma Chemical Co., and dihydro- ⁇ -erythroidine was a gift of Merck& Co.
• B waves B neurones
• C waves C neurones
• nAChR blockers namely d-tubocurarine and dihydro- ⁇ - erythroidine as well as ⁇ -conotoxin Ml from C. magus, which has been known for some time to block neuromuscular nAChRs (Olivera et al., 1985).
• the peak amplitudes of fast B waves and C waves were plotted as a function of time. Both B and C waves slowly declined over the 6.7 hour time span shown.
• the antagonists were ⁇ -conotoxin Iml (5 ⁇ M), ⁇ -conotoxin Ml (5 ⁇ M), d-tubocurarine (10 ⁇ M), ⁇ -conotoxin Mil (5 ⁇ M) and dihydro- ⁇ -erythroidine (5 ⁇ M), respectively. Representative response before, during and after exposure to each antagonist are shown in Figure 3. C waves were more readily blocked by Mil than were B waves.
• both fast and slow B waves were flocked equally well by ⁇ -conotoxin Mil; likewise, both components of C waves were equally sensitive to ⁇ -conotoxin Mil (see Figure 2D).
• 5 ⁇ M ⁇ -conotoxin Iml blocked the fast B wave by 82 ⁇ 5% and the fast C wave by only 14 ⁇ 12 %.
• Figure 3A shows that although ⁇ -conotoxin Iml's effect on the amplitude of the C wave was slight, it noticeably increased the latency of the response. The residual B wave's latency is also delayed.
• the figure also shows that the sensitivities of both fast and slow B waves to ⁇ -conotoxin Iml were very similar, as were the sensitivities of both components of the C wave.
• ⁇ -Conotoxin Ml is a well-established blocker of skeletal muscle nAChR's (Olivera et al., 1985).
• both ⁇ - conotoxins Iml and Mil are capable of blocking the nicotinic acetylcholine receptor of frog skeletal muscle (Mclntosh et al., 1994; Harris and Yoshikami, unpublished).
• Our working assumption is that these two ⁇ -conotoxins block synaptic transmission in frog ganglia by inhibiting nAChRs, although we have not rigorously ruled out other possibilities.
• ⁇ -conotoxin Ml indicates that the skeletal muscle subtype of nAChR does not play a significant role in synaptic transmission in sympathetic ganglia, a conclusion consistent with the observation that synaptic transmission in frog sympathetic ganglia, unlike that in skeletal muscle, is not blocked by ⁇ - Bungarotoxin (Shen et al., 1994).
• the simple method used in the present experiments of monitoring action potentials in the postganglionic nerve provides only an indirect assessment of an antagonist's ability to block nAChRs.
• the apparent potency of a nicotinic antagonist will depend on the "safety margin" of the excitatory synapse on the neurone; that is, how large the inward synaptic current is relative to that minimally necessary to reach threshold for generation of an action potential.
• a nicotinic antagonist would differentially block synaptic transmission through these two classes of neurones even if they possessed the same nAChRs.
• the intrinsic safety margins of the nicotinic synapses of B and C neurones would not be expected to be static but instead be dynamically influenced by pre- and post-synaptic modulation such as depression and facilitation (e.g., Shen & Horn, 1995), long-term potentiation (e.g., Minota et al., 1991), and the activities of homosynaptically-activated muscarinic receptors as well as hererosynaptically-activated peptidergic receptors (e.g., Jan and Jan, 1982; Kuffler & Sejnowski,
• pre- and post-synaptic modulation such as depression and facilitation (e.g., Shen & Horn, 1995), long-term potentiation (e.g., Minota et al., 1991), and the activities of homosynaptically-activated muscarinic receptors as well as hererosynaptically-activated peptidergic receptors (e.g
• a more quantitative, albeit considerably more involved, assay would be to voltage-clamp neurones and monitor spontaneous miniature epsc's (e.g., Thigpen, 1995) or the responses to exogenously applied ACh (e.g., Akaike et al., 1989). This would provide a direct measure of an antagonist's effect on AChR's. Use of such methods to characterize the toxins' effects remains for future experiments.
• the various nicotinic antagonists did not discriminate between fast and slow B waves, indicating that the same subtype of nAChR may be shared by all B neurones. Likewise, the two components of the C wave were not differentially blocked by the antagonists, suggesting that the neurones responsible for the two-component C wave all have the same subtype of nAChR.
• An increase in the latencies of partially blocked B and C waves was observed regardless of the antagonist producing the block (see Figure 3), and it is likely to be a reflection of an increased time required for the attenuated nicotinic epsp to reach threshold to generate the action potential.
• magus venom such as ⁇ -conotoxin Ml and ⁇ -conotoxin MVIIA, that paralyze fish by blocking neuromuscular transmission (Olivera et al., 1985).
• ⁇ -conotoxin Ml appears to be relatively ineffective in blocking ganglionic nAChRs.

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• 1997
  • 1997-11-17 CA CA002271769A patent/CA2271769A1/en not_active Abandoned
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