BR112015021380B1 - DOSAGE UNIT FOR THE TREATMENT OR PREVENTION OF PAIN IN AN AWAKE, SEDATE OR ANESTHESIA HUMAN BEING - Google Patents

Abstract

DOSAGE UNIT FOR THE TREATMENT OR PREVENTION OF PAIN IN AN AWAKE, SEDATE, OR ANESTHESIA HUMAN BEING AND METHOD OF USE. Since each of the site 1 sodium channel blockers has a unique activity and cannot be used to extrapolate the same effective dosage for another site 1 sodium channel blocker, studies were performed to identify neosaxitoxin dosages (" NeoSTX") and bupivacaine, alone or in combination with epinephrine, to provide two to three days of pain relief in humans. Bupivacaine-NeoSTX combinations produce more reliable block and longer duration block than NeoSTX alone. The three-way combination of NeoSTX-bupivacaine-epinephrine produces longer local anesthesia than the two-way combination of NeoSTX-bupivacaine. The addition of epinephrine to this NeoSTX-bupivacaine combination significantly prolongs the duration of complete blockade to a mechanical stimulus. These results led to the development of combination-specific dosage formulations.

Description

CROSS-REFERENCE TO RELATED ORDERS

[0001] This order claims priority to U.S.S.N. 61/789, 054 filed on March 15, 2013, entitled “Combinations of Neosaxoxixin with Bupivacaine and Epinephrine Increase Efficacy of Peripheral Nerve Blocks and Local Anesthesia and Infiltration Analgesia without Increasing Toxicity,” by Charles Berde, whose teachings are here incorporated.

FIELD OF THE INVENTION

[0002] This is usually in the field of improved nerve blocks and local infiltration anesthesia and analgesia without increased toxicity, specifically combinations of neosaxitoxin with bupivacaine, alone or in combination with epinephrine, in specific and total concentration dosages.

FUNDAMENTALS OF THE INVENTION

[0003] A non-sustained release agent that reliably gives 6 to 12 hours of surgical grade nerve block, followed by up to about 48 hours of lesser block and pain relief without further treatment is desirable. The first period would be useful with regard to the intraoperative period, as well as the immediate postoperative period; the latter would provide decreasing analgesia and allow increasing use of the body part involved as the healing progressed. ExparelTM, the only long-acting local anesthetic on the market, provides an unpredictable nerve block in humans that peaks at 24 h after injection, and the anesthetic effect is inversely proportional to dose. Furthermore, it involves the use of a sustained release system and causes local tissue damage and inflammation.

[0004] Similar considerations relate to bupivacaine + dexamethasone microparticles, which could provide local anesthesia for prolonged duration albeit with a sustained release system and with very severe tissue damage. QX-314, the quaternary lidocaine derivative, could provide long-lasting local anesthesia (about 24 h duration) but with very severe local tissue damage and systemic toxicity.

[0005] When amino-amide and amino-ester local anesthetics are given in overdose or by accidental intravascular injection, they generate cardiovascular toxicity that is notoriously refractory to resuscitation (Polaner et al. Ped Anes 2011;21:737-742; Fisher , et al., Can. J. Anaesth., 1997; 44: 592-598; Butterworth, Reg. Anesth. Pain Med., 2010; 35:167-76). The cardiovascular toxicity of bupivacaine is possibly mediated by the cardiac sodium channel Nav1,5 which is relatively more resistant to binding and inactivation by site 1 sodium channel blockers (Clarkson, et al., Anesthesiology, 1985; 62:396-405 ).

[0006] The phycotoxins neosaxitoxin, saxitoxin and goniauíatoxins are active compounds produced by harmful proliferations of algae of the genera Alexandrium sp., Piridinium sp., and Gimnodinium sp., (Lagos, N. Biol. Res., 31: 375-386 1998 )). In the last 15 years, it has been demonstrated that these phycotoxins can also be produced by freshwater cyanobacteria such as photosynthetic blue-green algae, in addition to being produced by marine dinoflagellates.

[0007] Only four genera of cyanobacteria capable of producing paralyzing phycotoxins have been identified, and each of them produces a different mixture of phycotoxins both in quantities and types of phycotoxins produced, that is, they produce different profiles of paralyzing phycotoxins (Lagos, et al. al., 1999, TOXICON, 37: 1359 - 1373 (1999) Pereira, et al., TOXICON, 38: 1689 - 1702 (2000).

[0008] The chemical structure of these phycotoxins has a general structure (I) and its particular structure is defined by the substituents R1 to R5 according to the following table:

[0009] These paralyzing phycotoxins act as a specific blocker of voltage-gated sodium channels present in excitable cells (Kao, C.Y., Pharm. Rev., 18: 997-1049 (1966)). Due to the inhibition of sodium channels, the transmission of a nerve impulse is blocked and the release of neurotransmitters is prevented at the level of the neuromotor junction, which prevents muscle contraction. Due to these physiological effects, these compounds are potentially useful in pharmacology, when used as inhibitors of muscle activity in pathologies associated with muscle hyperactivity, such as muscle spasms and focal dystonias, when applied locally in an injectable form. Furthermore, since a nerve impulse blockade at the transmission level is generated when these compounds are applied as a local infiltration, they are not only able to block the efferent pathways of neurotransmission, but also block afferent pathways and cause an inhibition of neurotransmission. sensory pathways and generate an anesthetic effect when these compounds are injected locally. This is a very interesting effect, since both effects are simultaneous, as described in Patent No. US 4,001,413.

[00010] As in the description of Patent No. US 6,326,020 to Kohane, et al., combinations of naturally occurring site 1 sodium channel blockers, such as tetrodotoxin (TTX), saxitoxin (STX), decarbamoyl saxitoxin, and neosaxitoxin, with other agents, have been developed to get long-lasting locks with improved features including security and specificity. In one embodiment, duration of blockade is greatly prolonged by combining a toxin with a local anesthetic, vasoconstrictor, glucocorticoid, and/or adrenergic drugs, both alpha agonists (epinephrine, phenylephrine), and mixed alpha-2-central peripheral agonists (clonidine), or other agents. Prolonged nerve block can be achieved using combinations of toxin with vanilloid. Dosage ranges based on tetrodotoxin and saxitoxin studies have been provided. However, it is now known that studies must be conducted with each toxin in order to predict effective dosages, as dosages with one type of toxin do not predict efficacy with another type of toxin. As demonstrated in the following examples, it has also been found that it is not possible to extrapolate from rats and sheep to humans to determine safe and effective dosages with respect to these toxins.

[00011] Conventional local anesthetics are associated with local neurotoxicity at clinical doses and profound cardiovascular toxicity at overdose. Although overall incidence is low, studies have also identified prolonged numbness and paresthesias as a complication of local and regional anesthesia with amide anesthetics. This has been associated with histological signs of chemical nerve damage (Myers, et al., Anesthesiology, 1986; 64:29-35; Kalichman, et al., J. Pharm. Exper. Therapeutics, 1989; 250(1): 406-413), These risks for local neurotoxicity are likely to be further increased in the setting where prolonged pain relief is attempted via administration of conventional local anesthetics by controlled-release delivery (Padera, et al. Anesthesiology, 2008; 108 : 921-8; Kohane and Langer, Chem. Sci., 2010; 1: 441-446) or local perineural infusions, particularly when higher concentrations or doses are used for extended periods of time. At equipotent doses injected intrathecally, site 1 sodium channel blockers cause longer duration of anesthesia with less histological evidence of neurotoxicity than bupivacaine (Sakura, et al., Anesth. Analg., 1995;81:338-46). In general, approaches to prolonged local anesthesia involving site 1 sodium channel blockers decrease the risks of nerve damage relative to approaches involving prolonged or recurrent administration of conventional amino amides or amino esters.

[00012] It is therefore an object of the present invention to provide specific combinations of neosaxitoxin with bupivacaine, optionally with epinephrine, to provide pain relief for up to two to three days after a single injection, which are both safe and effective in humans.

[00013] It is a further object of the present invention to provide formulations to provide safe and effective local anesthesia and analgesia in pediatric patients.

[00014] It is yet another object of the present invention to provide formulations for the treatment of indications that require high, medium and low volumes of local anesthesia.

[00015] It is a further object of the present invention to provide formulations for the treatment of indications that require different rates of recovery from sensory or motor block due to local anesthesia.

SUMMARY OF THE INVENTION

[00016] Studies have been conducted to identify dosages of neosaxitoxin ("NeoSTX") and bupivacaine, alone or in combination with epinephrine, to provide up to two to three days of pain relief. Studies using percutaneous sciatic nerve block in rats demonstrated that 1) Bupivacaine-NeoSTX combinations do not increase systemic toxicity compared to NeoSTX alone; 2) Bupivacaine-NeoSTX combinations produce more reliable block and longer duration block compared to NeoSTX alone; and 3) The three-way combination of NeoSTX-bupivacaine-epinephrine produces longer local anesthesia than the two-way combination of NeoSTX-bupivacaine. The addition of epinephrine to this NeoSTX-bupivacaine combination significantly prolongs the duration of complete block to a mechanical stimulus.

[00017] Studies in rats and sheep have shown that NeoSTX does not produce cardiotoxicity, even when injected intravenously. However, it is not possible to determine effective dosages in humans, adults or children, awake or awake, using animal studies, due to determinable limiting side effects in humans and not animals.

[00018] These two-way and three-way combinations use additive or synergistic interactions to increase efficacy, while the toxic effects of the combinations are less than additive, thus improving the margin of safety and overall efficacy. Based on these unexpected effects on efficacy, in rat and human studies, the chosen bupivacaine concentration, 0.2%, is lower than standard commercial bupivacaine concentrations of 0.25%, 0.5% or 0.75%.

[00019] The Human Phase 1 studies were conducted under an investigator-initiated IND with FDA and IRB approvals. The study demonstrated that combinations of NeoSTX-bupivacaine, alone or in combination with epinephrine, in different ratios for different clinical indications, provide a clinically important improvement in terms of efficacy, duration of pain relief, and safety for local anesthesia - compared to with the standard, bupivacaine. The results demonstrated: 1. 0.2% NeoSTX-bupivacaine combinations, even when NeoSTX doses are as low as 5-10 mcg (concentrations from 0.5 - 1 mcg/ml), and even without epinephrine , prolong the blockade in relation to the standard, bupivacaine, approximately 4 times (Figures 7 and 8). 2. NeoSTX alone, ie dissolved in normal saline, is inefficient and inconsistent at tolerable doses, so human data strongly support the need for the NeoSTX-bupivacaine combination (Figures 7 and 8) 3. Epinephrine reduces the systemic symptoms, thus widening the margin of safety, prolongs the duration of the dense block (suitable for surgical anesthesia) and prolongs the analgesic duration additionally (eg, from 48-72 hours), (Figure 8). This allows dosing in high volume applications which have recently become widely used for wound infiltration for hip and knee surgery (over 100 mls are routine) and major abdominal surgery. 4. Side effects (tingling, vomiting, nausea) limit the tolerable dose of NeoSTX-bupivacaine in awake patients to less than 40 mcg of NeoSTX, although higher doses should be safe in patients under general anesthesia. Note that these limiting side effects were seen with NeoSTX-saline and NeoSTX-bupivacaine, not with NeoSTX-bupivacaine-epinephrine.

[00020] Specific volumes and concentrations of components that will improve safety, tolerability, and efficacy in specific clinical situations have already been identified. These findings led to studies to develop the following combination dosage formulations:

[00021] A dosage formulation for high volume use of 35 - 120 ml for adult humans and 0.5 - 1.8 ml/kg for children, includes as the active agents a three-way combination of Bupivacaine in a range of concentration between 0.1% (1 mg/ml) and 0.25% (2.5 mg/ml), giving a total systemic dose of bupivacaine of no more than 225 mg in adults or 2.5 mg/kg in children; NeoSTX in a concentration range from 0.1 mcg/ml - 1 mcg/ml, giving a total systemic dose of 3.5 - 100 mcg in adults or 0.05 - 1.5 mcg/kg in children, and Epinephrine in a concentration range between 2 mcg/ml (1:500,000 in common terminology) and 10 mcg/ml (1:100,000). Common use of this formulation would be for three or four layer infiltration of a large surgical wound for a full open laparotomy, thoracoabdominal incision, or flank incision. Some of these operations include: Cesarean delivery, open hysterectomy, esophagogastrectomy, nephrectomy, or major abdominal cancer surgeries such as colectomy. Wound infiltration for total hip replacement (hip replacement) and total knee replacement (knee replacement) would also be ideal uses for these formulations.

[00022] Formulation of dosages for the use of an average volume of 15 to 50 ml, includes as active agents a combination of Bupivacaine in a concentration range of 0.125% - 0.3% (1.25-3 mg/ml) , giving a systemic dose in adults of no more than 100 mg (no more than 2 mg/kg in children), NeoSTX in a concentration range from 0.2 - 2 mcg/ml, giving a systemic dose in adults from 7 - 150 mcg (0.1 - 1.5 mcg/kg in children), and Epinephrine in a concentration range from 0 - 10 mcg/ml (< 1:100,000).

[00023] Many of the anticipated uses for moderate volume formulations involve both peripheral nerve blocks or plexus blocks (perineural injection) as well as infiltration (injection along the layers of a surgical wound). Uses of this formulation include shoulder, hand or arm surgery, infiltration or ilioinguinal/iliohypogastric blocks for inguinal hernia repair, penile block for hypospadias correction, femoral block for total knee replacement or anterior cruciate ligament repair, intercostal nerve blocks for open chest surgery, or femoral and sciatic nerve blocks for leg amputation. For hip surgery, this could involve lumbar plexus block and smaller volume sciatic block. This formulation can also be used for nerve blocks (femoral and sciatic, lumbar and sciatic plexus) for waist or knee joint replacement surgery.

[00024] For some of these medium volume uses, in particular with peripheral nerve blocks and plexus block, a high priority is to provide three resources: anesthesia (almost complete insensibility) for surgery, for periods of 3 -12 hours, analgesia (relief of prolonged pain) after surgery, for periods of at least 24 hours, while ensuring recovery from the motor block to allow some strength in limb movement for a period of time of 24 - 48 hours.

[00025] For peripheral nerve blocks and plexus blocks with motor effects in the arms and legs, based on the motor block recovery requirement of 24 - 48 hours, formulations with NeoSTX-bupivacaine, without epinephrine are likely to be ideal, as detailed in the following tables.

[00026] A dosage formulation for low volume and long duration, includes as the active agents a combination of Bupivacaine in a concentration of 0.25% - 0.5% (2.5 - 5 mg/ml), in which 5 - 15 ml dose gives a systemic bupivacaine dose in adults of no more than 75 mg, NeoSTX in a concentration range from 0.5 - 5 mcg/ml, where 5 - 15 ml dose gives one dose systemic in adults from 5 - 75 mcg, and Epinephrine in a concentration range from 2.5 - 10 mcg/ml (1:500,000 - 1:100,000). An example is lumbar sympathetic block for complex regional pain syndrome/reflex sympathetic dystrophy or vascular insufficiency of the leg or for celiac plexus block for pancreatitis or pancreatic cancer.

[00027] It is desirable to have this type of block lasting as long as possible, since when performed using fluoroscopic guidance in small volumes, there is very little of the sensory or motor block. Therefore, relatively high concentrations of all three components must be used for this application to achieve durations of sympathetic blockade and an increase in local blood flow for at least 3-4 days, and possibly longer. Other applications that may involve this long-term, low-volume use would include long-term sciatic nerve blocks where rapid motor recovery is not an issue, such as for a lower leg amputation.

BRIEF DESCRIPTION OF THE FIGURES

[00028] Figures 1A-1F show rat dose-response for sciatic nerve block intensity at 15 minutes (peak effect) with NeoSTX in saline and NeoSTX-bupivacaine. Figures 1A-1F are graphs of blockade intensity after injection into the sciatic nerve of rats as described in Example 1 at 15 minutes for Hotplate, EPT, and Von Frey tests in the injected hindlimb (Figures 1A-1C) and in the contralateral hind limb (Figure 1D-1F), comparing limb withdrawal latency (seconds) (1A, 1D); o Extensor postural impulse (g) (1B, 1E), and the force to withdraw the limb (g) (1C, 1F).

[00029] Figures 2A-2F show the duration of sciatic block in rats to NeoSTX in saline and NeoSTX-bupivacaine. Figures 2A-2F are graphs showing the duration of blockade after ischial injection in rats as described in Example 1 for the Hotplate recovery (hours) (2A, 2D), EPT recovery (hours) test (2B, 2E) , and Von Frey recovery (hours) (2C, 2E) in the injected hindlimb (2A-2C) and in the contralateral hindlimb (2D-2F).

[00030] Figure 3 shows that the addition of bupivacaine reduces the systemic toxicity of NeoSTX (increases the LD50). NeoSTX 4 and NeoSTX 5 refer to two different formulations prepared in different production facilities over a year apart. Figure 3 is a graph of LD50 curves (in micrograms) of 2 NeoSTX formulations, Neo 4 (lines on the left of the graph) and Neo 5 (lines on the right of the graph), with and without bupivacaine, based on data from rats .

[00031] Figures 4A-4C show that block durations are longer with NeoSTX-bupivacaine-epinephrine, intermediate with NeoSTX-bupivacaine, and shorter with bupivacaine alone. Figures 4A-4C are graphs of Von Frey test in rats over time for 0.2% bupivacaine (Figure 4A), 0.2% bupivacaine plus 3 µg NeoSTX/kg (Figure 4B), and 0 .2% bupivacaine added to 3 μg of NeoSTX/kg added to 5 μg of epinephrine/kg (Figure 4C).

[00032] Figures 5A and 5B show that the addition of Bupivacaine to NeoSTX improves the reliability and duration of the block compared to NeoSTX-Saline, as measured by rat sciatic nerve block. Addition of Epinephrine to NeoSTX-Bupivacaine produces further prolongation of blockade. The results demonstrate that NeoSTX in saline produces inconsistent and delayed onset of mechanical sensory block and block is fully recovered by 12 hours, even at a slightly higher NeoSTX dose (3.5 mcg/kg) than was used in Figure 5B (3 mcg/kg). Figures 5A and 5B are graphs of mechanical force application in grams in the Von Frey test over time in hours for the leg treated for 3.5 mcg NeoSTX (triangle) compared to control (circle) (Figure 5A) and how a function of 0.2% bupivacaine (circle), 0.2% bupivacaine and 3 mcg NeoSTX (square) or 0.2% bupivacaine 3 mcg NeoSTX 5 mcg epinephrine/ml (triangle) (Figure 5B). All three formulations produce rapid onset of dense mechanical sensory block. The graphs show that the addition of NeoSTX 3 mcg/kg to Bupivacaine 0.2% produces a 4-fold prolongation of total block (4 hours vs. hours). Results showed that the addition of 5 mcg/kg epinephrine to 0.2% Bupivacaine + 3 mcg/kg NeoSTX results in an additional 6-fold prolongation of complete block (24 hours versus 4 hours) and an additional 2.5-fold prolongation of half-maximal blockade (30 hours versus 12 hours) compared to Bupivacaine 0.2% + NeoSTX 3 mcg/kg. Compared to the current standard, bupivacaine, the 3-way combination of Bupivacaine-NeoSTX-Epinephrine, results in 24-fold prolongation of complete block and 15-fold prolongation of half-maximum blockade.

[00033] Figures 6A and 6B showed that systemic symptoms in the Phase 1 human study varied with NeoSTX dose, with symptoms being greatly suppressed by the inclusion of epinephrine. The addition of Epinephrine to NeoSTX-Bupivacaine combinations dramatically reduces the incidence and clinical relevance of systemic symptoms at NeoSTX doses of 10 mcg and 30 mcg in adult humans in the Phase 1 Trial. Figure 6A is a graph of the percentage of subjects having any systemic symptoms: tingling, numbness, dizziness, nausea or vomiting at any point in time after administration of 0, 5, 10, 15, 20, 30 or 40 mcg NeoSTX-bupivacaine-epinephrine. Nausea was observed in 80% of subjects at 40 mcg NeoSTX. The graph shows that the occurrence of systemic symptoms from any time point score for the NeoSTX-bupivacaine-epinephrine combination at NeoSTX doses of either 10 mcg or 30 met did not rise above placebo. Figure 6B is a graph of the percentage of subjects having clinically relevant systemic symptoms, ie, scores greater than 3 on a scale of 0-10 for 30 minutes or more. Figure 6B shows that there are no values greater than zero for NeoSTX-bupivacaine-epinephrine subjects receiving 10 mcg or 30 mcg doses.

[00034] Figure 7A and 7B show the effects of NeoSTX-Bupivacaine and NeoSTX-bupivacaine-epinephrine combinations on blockade intensity and duration in the Phase 1 human study using NeoSTX 10 mcg. Figures 7 A, B, and C are from the Phase 1 human Study showing that the addition of Epinephrine enhances and prolongs blockade of NeoSTX-Bupivacaine combinations. Figures 7A, 7B, and 7C are graphs of measurement of dense and partial block threshold, mechanical detection (Figure 7A), mechanical pain (Figure 7B), and cooling detection (Figure 7C) for NeoSTX, NeoSTX + bupivacaine, NeoSTX + bupivacaine + epinephrine compared to placebo and controls (without NeoSTX) over time in hours. The results demonstrate that Bupivacaine 0.2% causes dense blockade for no more than 6 hours, and partial analgesia between 6-12 hours. NeoSTX 10 mcg in saline gives rise to highly variable and short-lived blockade. NeoSTX 10 mcg in 0.2% Bupivacaine gives dense block for approximately 12 hours and degrees of analgesia for more than 24 - 72 hours. NeoSTX 10 mcg in 0.2% Bupivacaine with 5 mcg/ml epinephrine gives dense block for 24 hours and degrees of analgesia for more than 48 - 72 hours.

[00035] Figures 8A-8C show the effects of increasing NeoSTX Dose on 0.2% NeoSTX-bupivacaine Combinations. Figures 8A, B and C are from the Phase 1 Human Study showing that NeoSTX-Bupivacaine Combinations prolong blockade relative to bupivacaine alone at doses as low as 5 mcg. Figures 8A-C show graphs of dense and partial block measurement of mechanical (Figure 8A) and thermal (Figures 8B, 8C) detection for bupivacaine, NeoSTX-bupivacaine, and NeoSTX-bupivacaine-epinephrine at a NeoSTX dose of 10 mcg. Results show combinations of NeoSTX - Bupivacaine 0.2%, at all NeoSTX doses ranging from 5 mcg to 40 mcg, producing dense blockade of multiple sensory modalities or at least 12 hours, analgesia for periods of 24 - 72 hours , and reliable recovery from dense mechanical block within 48 hours, as required for use in peripheral nerve blocks that affect motor function in the arms and legs.

DETAILED DESCRIPTION OF THE INVENTION

[00036] The safety benefits of reducing bupivacaine dosage with NeoSTX are important for patients of all ages, but especially for children. Epidemiologic data from prospective registries indicate that younger children are at increased risk, compared with adults, of systemic reactions to local anesthetics. Local anesthetics and regional anesthesia are increasingly being used to provide pain relief after surgery in infants and children. The greater margin of safety offered by these combinations has a unique use in pediatrics. Optimal, preferred, and broad-range doses, volumes, and concentrations for pediatric patients for different indications were derived based on considerations related to the physicochemical properties of NeoSTX and how body compartment sizes and volumes of distribution dimension themselves. same with body weight in older children and infants. Definitions: Analgesia refers to insensitivity to pain without loss of consciousness. Anesthetic refers to a loss of sensation (local, not causing loss of consciousness; systemic, with loss of consciousness) and usually of consciousness without loss of vital functions. Vasoconstrictor is an agent for narrowing the lumen of blood vessels, especially as a result of vasomotor action. Infiltration refers to injection into multiple layers or areas of tissue. Injection refers to injection at a single point in the tissue or lumen. Nerve block refers to local anesthesia produced by interrupting the flow of impulses along a nerve trunk. Minimum effective concentration ("MEC") is the lowest local concentration of one or more drugs in a given location sufficient to provide pain relief.

IL Compositions A. Site 1 sodium channel blockers

[00037] Site 1 blockers are a family of molecules long recognized for their potent and specific blockade of voltage-gated sodium channels, Site 1 sodium channel blockers include tetrodotoxin (TTX), saxitoxin (STX), decarbamoyl saxitoxin , neosaxitoxin, and the goniautoxins (herein collectively referred to as "toxins"). Tetrodotoxins are obtained from the ovaries and eggs of several species of pufferfish and certain species of salamanders of the type Taricha torosa. Chemically, it is an amino perhydroquinoline. See Pharmacological Reviews, Vol. 18 No. 2, pp. 997-1049. Tetrodotoxin alone is too toxic to be used as an anesthetic. Combinations of tetrodotoxin with bupivacaine produced a long-lasting sciatic nerve block in rats without increased systemic toxicity compared with tetrodotoxin alone (Kohane, et al., Anesthesiology, 1998:119-131). Although the most widely known site 1 toxin, tetrodotoxin, is effective as an anesthetic, it is expensive for clinical use, since it must come from puffer fish; when the endosymbiotic bacteria that make TTX are cultured ex vivo, their production of TTX decreases.

[00038] Saxitoxin was first extracted from the Yellow Clam (Alaska butter ciam), Saxidomus giganteus, where algae of the genus Gonyaulax is present. The chemical formula reported is C10 H15 N7 O3. 2HCI. The toxin is believed to have a perhydropurine nucleus, in which two guanidinium radicals are incorporated. Saxitoxin is also too toxic to use alone as a local anesthetic.

[00039] Saxitoxin and its derivatives can be produced from algae in bioreactors. The two derivatives, neosaxitoxin (NeoSTX) and decarbamoyl saxitoxin, have advantages in terms of production process and potency. One study examined rat sciatic nerve block with various members of the saxitoxin series, including NeoSTX (Kohane, et al., Reg. Anesth. Pain Med., 2000;25:52-9). Saxitoxin and these two derivatives all give markedly synergistic and prolonged blockade (1-2 days in rat sciatic nerve in vivo) when combined with bupivacaine or epinephrine.

[00040] Neosaxitoxin and decarbamoyl saxitoxin are potentially more potent and may have advantages over saxitoxin in the formulation. Neosaxitoxin (NeoSTX) is under clinical development as a long-acting local anesthetic (Rodriguez-Navarro, et al., Anesthesiology, 2007;106:339-45; Rodriguez-Navarro, et al., Neurotox. Res., 2009;16 :408-15;Rodriguez-Navarro, et al., Reg. Anesth. Pain Med., 2011;36:103-9). A Phase 1 study of subcutaneous infiltration in human volunteers demonstrated that NeoSTX caused effective cutaneous hypoesthesia (Rodriguez-Navarro, et al., Anesthesiology, 2007; 106:339-45) and a second Phase 1 study demonstrated that combination with bupivacaine resulted in longer analgesia compared to NeoSTX or bupivacaine alone ( Rodriguez-Navarro, et al., Neurotox. Res., 2009; 16:408-15 ).

[00041] The preferred source of site 1 sodium channel blocker is neosaxitoxin produced by Proteus, Chile.

B. Local Anesthetics

[00042] As used herein, the term “local anesthetic”1 means a drug that provides local numbness or pain relief. Classes of local anesthetics that may be used include the aminoacylanilide compounds such as lidocaine, prilocaine, bupivacaine, mepivacaine and related local anesthetic compounds with various substituents on the amine ring or nitrogen system; aminoalkyl benzoate compounds such as procaine, chloroprocaine, propoxycaine, hexylcaine, tetracaine, cyclometicaine, benoxynate, butacaine, proparacaine, and related local anesthetic compounds; cocaine and related local anesthetic compounds; amino carbonate compounds such as diperodon and related local anesthetic compounds; N-phenylamidine compounds such as phenacaine and related anesthetic compounds; N-aminoalkyl amid compounds such as dibucaine and related local anesthetic compounds; aminoketone compounds such as phalicaine, dyclonine and related local anesthetic compounds; and amino ether compounds, such as pramoxine, dimethisoquien, and related local anesthetic compounds. The preferred local anesthetics are the amino esters and amino amides, with bupivacaine being the most preferred, the levoenantiomer of bupivacaine being preferred when the vasoconstrictor activity of the local anesthetic is desirable, tetracaine, and ropivacaine, which is slightly more sensory selective.

[00043] These drugs average six to ten hours of pain relief when administered at different sites and by different types of surgery. For many types of surgery, it would be preferable to have pain relief durations that last for two or three days. Preferred local anesthetics for use in combination with NeoSTX are bupivacaine, ropivacaine, tetracaine and levobupivacaine. Bupivacaine is a particularly long-acting and potent local anesthetic. Its other advantages include sufficient sensory anaesthesia, no partial motor block only, and wide availability.

C. Vasoconstrictors

[00044] Vasoconstrictors which are useful are those which act locally to restrict the flow of blood, and thereby retain the injected drugs in the region where they are administered. This has the effect of substantially decreasing systemic toxicity. Preferred vasoconstrictors are those that act on alpha-adrenergic receptors, such as adrenaline and phenyl-epinephrine. Other drugs and dyes cause vasoconstriction as a side effect, such as bupivacaine and levobupivacaine.

D. Excipients and Preservatives

[00045] The formulations may also contain preservatives, pH adjusting agents, antioxidants, and isotonicity agents.

[00046] In the preferred embodiment, the anesthetic is formulated in saline solution, or an acidic buffered solution, optionally containing a preservative. Local or sustained release vehicles can be used, but are not preferred.

E. Dosage Units

[00047] In preferred embodiments, NeoSTX, bupivacaine and epinephrine, optionally, are provided in vials in an aqueous solution. Depending on the type of formulation, as described above and below, vial sizes can range from 15 - 40 mL, and 1 - 3 vials can/can be used for a single patient in different situations. In another embodiment, NeoSTX, local anesthetic, and optionally vasoconstrictor, are provided in one or more vials, optionally lyophilized, then rehydrated and combined before use. For this second embodiment, preferred vial sizes can range from 5 - 40 mls,

lll. Formulations and Dosages

[00048] Animal and human studies are needed to determine effective dosages and volumes for treating humans. For example, the rank order of potency of site 1 sodium channel blockers from in vitro physiology experiments did not predict the order of potency of compounds in vivo (Kohane, et al., Reg. Anesth. Pain Med ., 2000; 25; 52-9).

[00049] The following principles impose limits on doses, volumes, and concentrations of local anesthetics.

[00050] Different clinical situations place different demands on the safety and efficacy of local anesthetics.

[00051] Systemic safety determines the upper limit of the total dose (mg or mg/kg) of bupivacaine, NeoSTX, or other local anesthetics. There are small differences in the total allowable dose based on the time course of absorption, vascularity, etc., but in general each local anesthetic has a maximum total allowable dose. As described herein, the inclusion of epinephrine along with NeoSTX and bupivacaine increases systemic efficacy and safety, allowing for higher doses or larger volumes and providing a longer duration of pain relief. However, there are some places in the body where epinephrine should be avoided due to excessive vasoconstriction. These include injections around the fingers and toes, and around the penis. Therefore, for different clinical uses, it is important to develop formulations with and without epinephrine.

[00052] In any localized region of the body, a sufficient concentration of local tissues of local anesthetics is required to block afferent transmission. The lowest local concentration of one or more drugs in a given location sufficient to provide pain relief is called the "Minimum effective concentration" or MEC.

[00053] Thus, clinical situations that require infiltration into large tissue volumes require larger total volumes of local anesthetic at or above the ECM than clinical situations that involve smaller tissue volumes. If the ECM is similar at different sites, then larger tissue volumes require a larger total dose than smaller tissue volumes.

[00054] For combinations of NeoSTX in saline and NeoSTX-Bupivacaine in the Phase 1 human clinical trial, it was interesting that systemic symptoms including mild tingling and/or numbness of the fingers, lips, and tongue occurred at doses of NeoSTX as as little as 10 mcg in total. These symptoms were mild at doses of 10 - 15 mcg, but increased in severity and were accompanied by transient nausea as doses were increased to 40 mcg. These symptoms were interesting because tingling and nausea were not knowable from non-human data. These human studies also showed that the addition of epinephrine to NeoSTX-Bupivacaine dramatically decreased the incidence of these symptoms, in the NeoSTX dose range from 10 - 30 mcg (see Figure 6). Therefore, there would be no way of knowing from previous data that epinephrine is needed at these concentrations to prevent tingling and nausea. In the Phase 1 human trial, epinephrine also dramatically increases the duration of NeoSTX+BPV blockade.

[00055] In some clinical situations, it is important not to give large volumes (ie greater than 20 ml) of local anesthetic in order to prevent the spread of numbness or weakness to other sites in the body where these effects are undesirable or even dangerous. For example, injection of volumes greater than 20 ml for an interscalene brachial plexus block, as used for shoulder surgery, increases the risk of spreading to nearby nerves in the neck that innervate the larynx, creating hoarseness and risk for aspiration of liquids into the trachea. Similarly, volumes greater than 20 ml for lumbar sympathetic block increase the risk of spillover to somatic nerves, with an associated risk of temporary leg weakness due to numbing of nerves to major leg muscles. In other settings, unless large volumes are used, some tissues are overlooked, and inadequate pain relief will result. Examples of these are cited below.

[00056] Human clinical trials have uncovered specific dosing considerations that were not predictable based on what was previously known about site 1 sodium channel blockers. Some of these are contrary to conventional wisdom and current clinical practice in local/regional anesthesia . These considerations further impact the specific formulations that can be used safely in humans in a way that was not anticipated.

[00057] In rats, detectable nerve block begins at more than 30 µM in 0.1 ml of injected material (Kohane, et al., RAPM, 25(1):1-107 (2000)), which corresponds to a dose of approximately 1 μg in a mouse weighing 350 g, which corresponds to a dose of approximately 270 μg in a human weighing 70 kg. As described in Example 1 and in additional studies, dose response was determined in rats for local anesthesia using NeoSTX in saline, NeoSTX-bupivacaine, and NeoSTX-bupivacaine-epinephrine (see Figures 1, 2, 4, and 5) . Bupivacaine concentration of 0.2% was the same for both rat and human studies. In rats, NeoSTX-Bupivacaine Combinations gave inconsistent or statistically insignificant prolongation of blockade compared to bupivacaine alone at NeoSTX Doses of 2 mcg/kg, and reliable and robust prolongation was only achieved at NeoSTX Doses of at least 3 mcg/kg . These studies were performed using an injection volume of 0.3 ml. Based on rat weights (about 250 mg), for these injections, NeoSTX Doses of 3 mcg/kg corresponded to NeoSTX Concentrations in the injected materials of 2.5 mcg/ml.

[00058] In contrast, in the Phase 1 human trial using NeoSTX-Bupivacaine Combinations, NeoSTX doses as low as 5 mcg (approximately 0.07 mcg/kg for these adult humans) were found to give an excellent, or it is. 4-fold greater blockade prolongation compared to bupivacaine alone (Figure 7), For an injection volume of 10 mls, this indicates a very strong blockade-prolonging effect for NeoSTX in NeoSTX-Bupivacaine combinations in humans using a NeoSTX concentration of 5 mcg/10 mis, ie. 0.5 mcg/ml.

[00059] Thus, NeoSTX in humans produces reliable prolongation of blockade in NeoSTX-Bupivacaine combinations at a 40-fold weight-based dose of NeoSTX at a concentration of NeoSTX at least 5-fold lower than corresponding doses and concentrations of Effective and weight-based NeoSTX found in rats. The schedule for drug dosing and hence specific ranges of concentrations claimed for specific types of surgeries could not be derived from the prior art because animals cannot be interrogated for symptoms that do not elicit very significant toxicity. Specifically, it has been found in human studies of NeoSTX that systemic symptoms including tingling, numbness of the lips, fingers and tongue, and eventually nausea occur at a weight-scaled dosage (15 - 40 mcg, or approximately 0.2 - 0 .6 mcg/kg) much lower than that used in rat studies (2-5 mcg/kg), see Figures 1 - 5, tingling, numbness and nausea are encountered both with impending toxicity from existing local anesthetics and from paralyzing fruit intoxication from the sea.

In summary:

[00060] The occurrence of these symptoms at these low doses was not anticipated by previous studies in rats or humans using NeoSTX.

[00061] For NeoSTX, symptoms occur far below doses that produce measurable physiological effects, such as weakness or impaired respiratory measurements. These symptoms limit the maximum doses of NeoSTX-Bupivacaine in awake subjects more than in anesthetized subjects.

[00062] NeoSTX symptoms carry less risk than when similar symptoms occur for bupivacaine overdose. If bupivacaine causes a tingling, odds are a person will soon either have a seizure, or arrhythmia, or cardiac arrest. This is not the case for NeoSTX.

[00063] Epinephrine dramatically reduces the severity of these symptoms, allowing tolerability of higher doses of NeoSTX in these 3-way combinations compared to the 2-way combinations.

[00064] The schedule for drug dosage and, consequently, the specific ranges of concentrations claimed for specific types of surgery is contrary to what is practiced worldwide in the dosage of local anesthetics. Systemic toxicity is rarely a limiting factor in the use of conventional local anesthetics, so concentration adjustment to account for the volume of material injected is not necessary. The use of NeoSTX, and similar compounds, requires a different approach, where a change in the composition of the drug solution itself is required.

[00065] Optimized formulations for different types of clinical situations were developed.

"High volume infiltration analgesia".

Tabela 2: Formulações de Volume Grande (Crianças) (Todas as listagens de 7kg” referem-se a um redimensionamento do peso corporal da criança)

[00066] A dosage formulation for high volume use of 35 - 120 ml for adult humans and 0.5 - 1.8 ml/kg for children, includes as the active agents a three-way combination of Bupivacaine in a range of concentration between 0.1% (1 mg/ml) and 0.25% (2.5 mg/ml), giving a total systemic dose of bupivacaine of no more than 225 mg in adults or 2.5 mg/kg in children; NeoSTX in a concentration range from 0.1 mcg/ml - 1 mcg/ml, giving a total systemic dose of 3.5 - 100 mcg in adults or 0.05 - 1.5 mcg/kg in children, and Epinephrine in a concentration range between 2 mcg/ml (1:500,000 in common terminology) and 10 mcg/ml (1:100,000). Common use of this formulation would be for three or four layer infiltration of a large surgical wound for a full open laparotomy, thoracoabdominal incision, or flank incision. Some of these operations include: Cesarean delivery, open hysterectomy, esophagogastrectomy, nephrectomy, or major abdominal cancer surgeries such as colectomy. Wound infiltration for total hip replacement (hip replacement) and total knee replacement (knee replacement) would also be ideal uses for these formulations. Common use of this formulation is for three- or four-layer infiltration of the surgical wound for a full open laparotomy, thoracoabdominal incision, or flank incision. Table 1: Large Volume Formulations (Adults)

Table 2: Large Volume Formulations (Children) (All 7kg” listings refer to a scaling of the child's body weight)

"Average volume and intermediate duration".

[00067] These uses involve a total volume of approximately 15-40 ml in adults or 0.2 - 0.6 ml/kg in children. In these applications, the volume must be kept relatively low to avoid spillage to undesirable targets. For example, for shoulder surgery, interscalene brachial plexus block is commonly used. Ultrasound guidance is used for precise needle placement, and the overall volume is limited to prevent squeezing into undesirable targets such as the recurrent laryngeal nerve (which affects laryngeal function). Blocks currently available usually last 10 hours and rarely up to 15-18 hours. This usually means that the blockages last well into the night, and patients have severe pain the first postoperative night.

[00068] In this regimen, it is desirable to have blocks reliably producing pain relief for at least 24 hours (i.e. to get through the first night), and up to 48 - 72 hours, but dense motor block and touch components The smooth onset of the block should subside in about 24 hours so that the patient has greater mobility in the arm and hand (for brachial plexus blocks) or leg (for femoral, lumbar plexus, or sciatic blocks) for the day after the surgery.

[00069] Formulation of dosages for the use of an average volume of 15 to 50 ml, includes as active agents a combination of Bupivacaine in a concentration range of 0.125% - 0.3% (1.25 - 3 mg/ml) , giving a systemic dose in adults of no more than 150 mg (no more than 2 mg/kg in children), NeoSTX in a concentration range from 0.2 - 2 mcg/ml, giving a systemic dose in adults from 7 - 100 mcg (0.1 - 1.5 mcg/kg in children), and Epinephrine in a concentration range from 0 - 10 mcg/ml (< 1:100,000).

[00070] Many of the anticipated uses for moderate volume formulations involve both peripheral nerve blocks or plexus blocks (perineural injection) as well as infiltration (injection along the layers of a surgical wound). Uses of this formulation include shoulder, hand or arm surgery, infiltration or ilioinguinal/iliohypogastric blocks for inguinal hernia repair, penile block for hypospadias correction, femoral block for total knee replacement or anterior cruciate ligament repair, intercostal nerve blocks for open chest surgery, or femoral and sciatic nerve blocks for leg amputation or foot and ankle surgery. For hip surgery, this could involve lumbar plexus block and smaller volume sciatic block. This formulation can also be used for nerve blocks (femoral and sciatic, lumbar and sciatic plexus) for waist or knee joint replacement surgery.

[00071] For some of these mid-volume uses, in particular with peripheral nerve blocks and plexus blocks, a high priority is to provide three features:

[00072] anesthesia (almost complete numbness) for surgery, for periods of 3 - 12 hours, analgesia (prolonged pain relief) after surgery, for periods of at least 24 hours, while ensuring recovery from motor block to allow some strength in limb movement for a period of time of 24 - 48 hours

Tabela 4: Formulações de Volume Médio (Crianças) (Todas as listagens de 7kg” referem-se a um redimensionamento do peso corporal da criança)

[00073] For peripheral nerve blocks and plexus blocks with motor effects in the arms and legs, based on the motor block recovery requirement of 24 - 48 hours, formulations with NeoSTX-bupivacaine, without epinephrine are likely to be ideal, as detailed in the following tables. Table 3: Medium Volume Formulations (Adults)

Table 4: Medium Volume Formulations (Children) (All 7kg” listings refer to a scaling of the child's body weight)

'Low volume and long duration'

[00074] This formulation is for sites where very prolonged effect is desirable, and where volumes can be kept small to avoid spillover to other sites. An example is lumbar sympathetic block for complex regional pain syndrome/reflex sympathetic dystrophy or vascular insufficiency of the leg or for celiac plexus block for pancreatitis or pancreatic cancer.

[00075] For lumbar sympathetic block, the injection is performed to block a group of nerves that produce vasoconstriction in the leg. When these nerves are blocked, the result is increased blood flow to the leg, and a reduction in pain from certain ailments. For this nerve block, volume should be relatively low (8-20 mls preferred) to avoid spillover to the somatic nerves of the lumbar plexus, which would make the leg weak. However, as opposed to medium volume and intermediate duration, it is desirable to have this type of block last as long as possible, since when performed using fluoroscopic guidance in small volumes, there is very little of the sensory or motor block. Therefore, relatively high concentrations of all three components must be used for this application to achieve durations of sympathetic blockade and an increase in local blood flow for at least four days.

[00076] A dosage formulation for low volume and long duration, includes as the active agents a combination of Bupivacaine in a concentration of 0.25% - 0.5% (2.5 - 5 mg/ml), in which 5 - 15 ml dose gives a systemic bupivacaine dose in adults of no more than 75 mg, NeoSTX in a concentration range from 0.5 - 5 mcg/ml, where 5 - 15 ml dose gives one dose systemic in adults from 5 - 75 mcg, and Epinephrine in a concentration range from 2.5 - 10 mcg/ml (1:500,000 - 1:100,000). An example is lumbar sympathetic block for complex regional pain syndrome/reflex sympathetic dystrophy or vascular insufficiency of the leg or for celiac plexus block for pancreatitis or pancreatic cancer.

Tabela 6: Formulações de Volume Baixo (Crianças)

Combinações de Duas Drogas de NeoSTX + BPV[00077] It is desirable to have this type of block lasting as long as possible, since when performed using fluoroscopic guidance in small volumes, there is very little of the sensory or motor block. Therefore, relatively high concentrations of all three components must be used for this application to achieve durations of sympathetic blockade and an increase in local blood flow for at least 3 - 4 days, and possibly longer. Other applications that may involve this low volume, long duration use would include extended duration sciatic nerve block where rapid motor recovery is not an issue, such as for a lower leg amputation. Table 5: Low Volume Formulations (Adults)

Table 6: Low Volume Formulations (Children)

Two Drug Combinations of NeoSTX + BPV

[00078] It is desirable to have a non-sustained release agent that reliably gives 6-12 hours of surgical grade nerve block, followed by up to about 48 hours of lesser block without further treatment. The former period would be useful intraoperatively as well as in the immediate postoperative period; the latter would provide decreasing analgesia and allow increasing use of the involved body part as healing progressed. NeoSTX + BPV can produce this block duration.

[00079] The present invention will be better understood by reference to the following non-limiting examples.

Example 1: Neosaxitoxin (NeoSTX) with bupivacaine provides long-lasting local analgesia in clinical trials without an increase in toxicity.

[00080] A recent study of NeoSTX in sheep using subcutaneous injection demonstrated that bupivacaine did not worsen surrogate measures of neuromuscular or respiratory toxicity of NeoSTX. In a separate model, deliberate intravenous infusion of NeoSTX showed remarkably mild cardiovascular toxicity, much lower than in previous comparable studies of bupivacaine. The present study further investigates the dose response of NeoSTX and NeoSTX-Bupivacaine combinations on neurobehavioral measures of rat sciatic nerve block, as well as the local and systemic toxicities of these combinations. These experiments were performed as a preclinical study for an Investigational New Drug Application, using NeoSTX formulations manufactured for clinical use in a planned Phase 1 clinical trial.

[00081] The hypotheses were the following: 1) at fixed NeoSTX doses, addition of bupivacaine increases the intensity and duration of rat sciatic nerve block; 2) in the presence or absence of bupivacaine, increases the intensity and duration of blockade with a dose of NeoSTX; 3) the histological effects of NeoSTX (in saline or in combination with bupivacaine) on rat sciatic nerve are benign across the intended dose range, and not statistically different from those of vehicle or untreated nerves; 4) in a model of rapid accidental intravenous infusion, NeoSTX and bupivacaine separately generated respiratory and electrocardiographic parameter points with different time courses. Combinations using full concentrations of both NeoSTX and bupivacaine developed systemic toxicity more rapidly (i.e., with shorter infusion times and lower cumulative dose), whereas combinations of half concentrations of each component developed toxicity more slowly, i.e., with a higher cumulative dose.

Materials and methods

[00082] Methods: NeoSTX, 0.25% bupivacaine, or combination was given via injection into the sciatic nerve of Sprague-Dawley rats. Sensory-nocifensive function was assessed by Hotplate and Von Frey filaments tests. Motor-proprioceptive function was assessed by Extensor Postural Impulse. Seven days later, sciatic nerves were dissected and examined histologically for toxicity. LD50 was also calculated for combination of NeoSTX and NeoSTX-Bupivacaine after sciatic injection. To model accidental intravenous overdose, isoflurane-anesthetized, spontaneously breathing rats received infusions of either NeoSTX alone, bupivacaine alone, or NeoSTX-Bupivacaine Combinations until they reached respiratory and electrocardiographic endpoints.

drugs

[00083] In the sciatic nerve injection model, the drugs were prepared on the day of the experiment and the volume of injected material was fixed at 0.3mL NeoSTX (Proteus SA, Chile) was transported and stored in accordance with the safety standards of the Children's Hospital Boston, in accordance with the Harvard Committee on Microbiological Safety. Standard NeoSTX contains 20mcg/mL of neosaxitoxin at pH 4.5.

[00084] NeoSTX was diluted in 0.9% saline or bupivacaine hydrochloride (SENSORCAINE®, APP Pharmaceuticals, Schaumberg, IL). Depending on the intended final NeoSTX concentration, commercial vials of bupivacaine such as bupivacaine 5 mg/ml (0.5% or bupivacaine 2.5 mg/ml (0.25%) were used to achieve final bupivacaine concentrations of 2 mg/ml ml (0.2%) in the final injected materials. In the IV overdose model, infusion concentrations were as follows: bupivacaine 2mg/ml, NeoSTX 1.88 mcg/ml, full concentration combination half concentration combination 2 mg/ml and NeoSTX 1.88 mcg/ml, half concentration combination: bupivacaine 1 mg/ml and NeoSTX 0.94 mcg/ml. Infusion rates were adjusted according to animal weight to ensure delivery of constant delivery rates of drugs scaled by weight. Thus, as with individual drugs, bupivacaine was administered at 3.2 mg/kg/min and NeoSTX at 3 mcg/kg/min. Full-dose combination animals both received bupivacaine 3.2 mg/kg/min and NeoSTX at 3 mcg/kg/min, while half-dose combination animals received bupivacaine at 1.6 mg/kg/min and NeoSTX 1.5 mcg/kg/min), and all animals received a constant rate fluid delivery rate scaled based on weight of 1.6 ml/kg/min.

Care for the Animals

[00085] Male Sprague-Dawley rats were obtained from Charles River Laboratories (Wilmington, MA): Young adults weighing 200g to 250g were used for the sciatica injection model and animals weighing 325-400g for the injection model. IV overdose. Animals were cared for and euthanized in accordance with protocols approved by the Institutional Animal Care and Use Committee at Children's Hospital. Handling procedures were developed to habituate the animals to the test paradigm and minimize stress-induced analgesia.

Sciatica Injection

[00086] Rats were briefly anesthetized with isoflurane by a nose cone. The needle was introduced posteromedial to the greater trochanter, pointing in an anteromedial direction. Once bone was contacted, 0.3 ml of solution was injected. The left leg was always used for blocks; the right served as a control and measure of systemic toxicity (Kohane, et al., Anesthesiology, 1998:119-131).

neurobehavioral test

[00087] A modified neurobehavioral assessment battery from Thalhammer et al. which employs sensory-nocifensive and motor-proprioceptive impairment measures to assess blockade duration and intensity after sciatic perineural injection (Thalhammer, et al., Anesthesiology, 1995;82:1013-1025). Investigators were blinded to dose and treatment assignment. Sensation was first assessed using Von Frey filament (VF) tests (Touch-Test Sensory Evaluator, North Coast Medical Inc., CA). After brief habituation to a wire mesh cage, upward force Von Frey filaments were applied until paw withdrawal was observed. Care was taken to apply filaments only to lateral plantar surfaces receiving reliable innervation from the sciatic nerve. The filaments were applied in an increasing series until withdrawal was observed or until a maximum of 300g was reached (Yahalom, et al., Anesthesiology, 2011;114(6): 1325-1335). The Extensor Postural Thrust (EPT) strength in grams exerted on a scale and the removal time of the Hotplate set set at 56 degrees Celsius were measured, in accordance with what was described in Kohane, et al. (Kohane 2000)

[00088] Neurobehavioral measurements were taken at pre-injection baseline, 15 minutes, 1, 2, 3, 4, 6, 8, 10, and 12h on the first day and then every 4h on the second day until recovery motor. At each time point, three measurements of EPT strength and Hotplate latency were performed and averaged. Previous studies of site 1 sodium blockers using this paradigm have shown that higher doses caused transient contralateral impairments of neurobehavioral measures, reflecting systemic analgesia and/or systemic weakness (Kohane, et al., Reg. Anesth. Pain Med., 2001; 26(3)239-45).

[00089] In previous studies, injections with 0.3 ml of bupivacaine 2.5 mg/ml resulted in complete block (based on breakpoints defined later in this paragraph) for > 98% of animals. In the analysis, the assumption was made that incomplete blockade with trial formulations reflects the true pharmacological effects of that dose, rather than a technical failure of the injection. Previous work used cutoff points to rate animals in achieving a sensory or motor block: 7s for Thermal Latency, 40g for Extensor Postural Impulse. 50% recovery was defined as 7s, and if the cutoff was not reached, then block duration was considered zero. It was found that the use of these cutoff points can discount degrees of sensory and motor impairment from potential clinical significance. To refine these cutoffs, the greatest change from baseline at 15 minutes after vehicle injection was calculated and these values used as the threshold for a Hotplate and EPT block (0.8s and 8g, respectively). Blocking limit was considered to be 60 g for the Von Frey test. Recovery was defined as return to the same values.

Histological procedures

[00090] Seven days after the sciatic injection, the rats received an overdose of pentobarbital (150 mg/kg) and were fixed by transcardial perfusion in two stages: 100 mL of 0.9% saline was infused, followed by 200 mL of a modified Karnovsky fixative containing 2.5% glutaraldehyde and 1.25% paraformaldehyde in 0.1 M phosphate buffer. Left and right sciatic nerves were dissected and stored in diluted fixative at 4 °C. Sciatic tissue was encapsulated in plastic using standard osmium tetroxide electron microscopy protocol, cut into semi-thin sections and stained with toluidine blue. Sections were analyzed by an experienced neurologist (G.C.), using the Estebe & Myers scoring system; this neuroscientist remained blind to group assignments throughout (Estebe, Anesthesiology, 2004; 100:1519-25).

Systemic Toxicity with Perineural Sciatica Injection.

[00091] Sublethal systemic toxicity was assessed by measuring sensory-nocifensive and motor-proprioceptive impairments of the right hindlimbs after sciatica injections of the left limb, as described in the above paragraphs under “Neurobehavioral testing”. At higher doses of NeoSTX, alone or in combination with bupivacaine, an increasing number of animals developed apnea or wheezing. To minimize suffering in this paradigm involving awake animals, any animal developing apnea or panting was immediately euthanized with intraperitoneal pentobarbital (100 mg/kg), and this was taken as a lethal event. Calculation of the LD50 is described in the Statistical Procedures section below.

Systemic toxicity with intravenous infusion

[00092] To model an accidental IV injection, spontaneously breathing rats anesthetized with isoflurane received infusions via tail vein cannula of a solution containing the drug until the endpoint of asystole. 26 rats were randomly divided into 4 groups: plain NeoSTX (n = 6); Bupivacaine (n = 7); NeoSTX-Bupivacaine combination at full concentration (n = 7); and NeoSTX Bupivacaine Combination in half concentration (n = 6), using the drug concentrations and infusion rates detailed in the section entitled “Drugs" above. Anesthesia was induced by inhalation of 3-5% isoflurane in oxygen and maintained for 1% isoflurane through a nose cone.A catheter was placed in the tail vein, flushed with 2 mL of 0.9% saline and connected to a Medfusion syringe pump (Smiths Medical, St Paul, MN). Vital vitals were monitored and data acquired continuously physiological using Powerlab equipment and LabChart software (AD Instruments, Sydney, Australia). Baseline measurements are taken (subsequent offline analysis) once all monitoring equipment is calibrated and connected (ECG, temperature , pulse oximeter, Bain circuit pressure transducer, and tail vein plethysmography), tail vein accessed, and the rat maintained in a stable plane of anesthesia in 1% isoflurane inspired oxygen for at least 5 minutes. Infusions as described in the paragraph titled "Drugs" above were initiated immediately after a short baseline recording period and continued until asystole was achieved. Primary endpoints for analysis were as follows: (1) apnea (pressure changes undetectable on Bain's circuit), and (2) asystole. Secondary endpoints were: bradycardia (heart rate <270), deleterious change in electrocardiogram waveform (including any heart block, wide QRS complex, ectopic atrial beats, or or prolonged QT interval), and loss of pulsatility of the caudal artery by plethysmography.

Statistical Procedures

[00093] All measurements are summarized as medians with interquartile ranges or mean ± standard deviation (mean ± SD) of the mean. Changes in tests of neurobehavioral function were evaluated in a nonparametric Friedman model with treatment and dose of NeoSTX with fixed factors with Bonferroni-adjusted P values in multiple comparisons (Montgomery, D., Design and Analysis of Experiments, 5th Ed. 2001, New York, NY: John Wiley & Sons, Inc.). Clinically relevant dose combinations of 1 or 2 mcg/kg were further explored with Mann-Whitney U tests. Nerve histology was analyzed with a Kruskal-Wallis model. Probit analysis using maximum likelihood was applied to calculate the mean lethal dose (LD50) for each drug treatment with a 95% likelihood ratio confidence intervals obtained by the profile log-likelihood method in the IV overdose model, time to event were summarized using Kaplan-Meier curves. Several pairwise comparisons of survival curves to Bupivacaine alone were conducted and P values of less than 0.017 were found to be statistically significant (Finney, Arch. Toxicol., 1985; 56:215-218; Faraggi, et al., Statist. Med. , 2003; 22:1977-1988). Statistical analysis was performed using the SPSS statistical package (version 19.0, SPSS Inc./IBM, Chicago, IL).

Results:

[00094] Over a range of doses, addition of bupivacaine to NeoSTX caused more intense and more prolonged blockade of nocifensive and motor-proprioceptive function compared to NeoSTX alone. See Table 7. Overall histologic lesion scores were very low for all groups, with median and IQR values of 0 on an Estebe-Myers scale. With subcutaneous injection, addition of bupivacaine to NeoSTX did not produce an increase in systemic toxicity (LD50) compared to NeoSTX alone. With intravenous infusion, NeoSTX, bupivacaine, and combinations showed different time courses to reach respiratory versus electrocardiographic parameter points. See Table 8.

neurobehavioral measures Blocking Intensity

[00095] Figures 1A-1F show the dependence of the intensity of the blockade in 15 minutes with the dose of NeoSTX administered in the presence or absence of bupivacaine. All bupivacaine-containing formulations were associated with complete blockade of all three behavioral measures at that time point. In the absence of bupivacaine, doses of NeoSTX alone equal to or less than 3 mcg/kg produced incomplete blockade in most animals.

Block Duration

[00096] Compared to animals that received injections of NeoSTX in saline, addition of bupivacaine produced substantial increases in time to recovery from 100% thermal and mechanical sensory-nocifensive and motor-proprioceptive blockade (P < 0.001). Results are shown in Figures 2A-2F Paired analysis with Bonferroni correction of groups receiving increasing doses of NeoSTX with and without bupivacaine demonstrated that the bupivacaine group had significantly longer block durations at all doses greater than 1 mcg/kg ( Figure 2A).

Os valores são a média (IQR).[00097] The mean time to 100% recovery after injection of 0.25% bupivacaine alone was 2.2h (1.9 - 2.9) for Hotplate test, 2.2h (1.8 - 2, 6) for EPT test, and 1.9h (1.7 - 2.7) for VF test. Injection of 0.25% bupivacaine combined with 1 mcg/kg of NeoSTX provided significantly increased time to 100% Von Frey recovery (2.8h, 2.2 - 3.5, P = 0.05) and tests of EPT (3.1h, 2.8 - 3.9, P < 0.001), but did not increase time to 100% recovery from harmful hotplate behavior (2.5h, 2.0 - 3.5, P = 0 ,4). Time to 50% recovery, in hours, was calculated for all tests and doses and is shown in Table 7. Time to 50% Von Frey recovery was significantly increased in animals after 1 mcg/kg NeoSTX and bupivacaine compared with bupivacaine alone (2.5h, 1.7 - 2.9 compared with 1.5h, 1.5 - 2.1, P = 0.03) whereas EPT and hotplate were not significantly different. However, the combination of 2mcg/kg of NeoSTX with bupivacaine elicited a significant and substantially greater increase in time to recovery from 100% hotplate response (10.8h, 9.1 - 17.8, P < 0.001), EPT response (22h , 15 - 28, P < 0.001), and Von Frey response (4.7h, 3 - 11, P < 0.001). Table 7: Time to 50% recovery of injected limb by hotplate, EPT, and Von Frey test.

Values are the mean (IQR).

Systemic toxicity

[00098] As a marker for systemic drug distribution after sciatic injection, neurobehavioral measurements were obtained from the uninjected right limb. Compared with bupivacaine alone, injection of NeoSTX with bupivacaine was associated with increased intensity of EPT blockade at 15 minutes (P = 0.001), but was not significantly associated with changes in hotplate or Von Frey response (Figures 2C, 2E ). In the Bonferroni model corrected using NeoSTX dose, and the presence of bupivacaine as variables, combination of NeoSTX with bupivacaine produced significantly greater hotplate (2A, 2D) and EPT (2B, 2E) blockade than bupivacaine alone in the contralateral limb at doses of 3 mcg/kg (P = 0.011 and P = 0.038, respectively) and 3.5 mcg/kg (P < 0.001 and P = 0.036, respectively). For the relatively small contralateral blocks observed, ANOVA did not reveal significant differences in time to 100% recovery in the contralateral limb between NeoSTX and NeoSTX-Bupivacaine combination animals when NeoSTX dose was less than 3 mcg/kg (Figure 2E).

LD50 test

[00099] Given the lack of mortality in the 3 mcg/kg NeoSTX group, the dosage was increased to a maximum of 8 mcg/kg. This maximum dose produced 100% mortality within 30 minutes of administration, for both NeoSTX alone and in combination with bupivacaine. In all animals, death was due to terminal apnoea. LD50 was calculated at 4.9 mcg/kg (95% Cl = 4.2 - 6.2) for NeoSTX alone and 5.7 mcg/kg (95% Cl = 4.9 - 7.9) for NeoSTX with Bupivacaine (Figure 4). NeoSTX escalation across the measured range was significantly associated with increased lethality (Z = 5.82, P < 0.001) and the mortality-decreasing effect of adding bupivacaine approached significance (Z = 1.86, P = 0.06). The point of emphasis here is that co-administration of bupivacaine decreased, rather than increased, the systemic toxicity of NeoSTX. Table 8: Baseline vital signs for IV overdose model across 4 Treatment Groups (n = 26).

Histology of Nerves

[000100] The Estebe-Myers score of nerve damage revealed a very benign histological profile after sciatic injection. For all treatment conditions, the median Estebe-Myers nerve injury score was 0 (IQR 0-0). No nerves were evaluated at a score of 3 or 4. There were no statistical differences between any sciatic nerves of treatment group and uninjected control (right), the total numbers of nerves examined included the following: vehicle 19, bupivacaine 0.25% single 19, contralateral (right side not injected) 16, NeoSTX 1 mcg/kg in saline 4, NeoSTX 1 mcg/kg in bupivacaine 8, NeoSTX 2 mcg/kg in saline 4, NeoSTX 2 mcg/kg in bupivacaine 7, NeoSTX 3 mcg/kg in saline 19, NeoSTX 3 mcg/kg in bupivacaine 27, NeoSTX 3.5 mcg/kg in saline 12, NeoSTX 3.5 mcg/kg in bupivacaine 13, NeoSTX 4 mcg/kg in saline 1, NeoSTX 4 mcg/kg in bupivacaine 6. As a validation check on blinded histologist readings, slides were obtained from positive control nerve sections harvested from animals that received deliberate nerve injury (loose ligation model ), processed under the same protocol. These positive control nerves all received high injury ratings, with Estebe-Myers scores of 3 or 4.

[000101] This demonstrates that the combination of NeoSTX with bupivacaine increases the duration and reliability of the sciatic nerve block without increasing neurotoxicity or increasing systemic toxicity (reducing LD50) after subcutaneous injection.

[000102] The study demonstrates that, in rats, ischial co-injection of 0.25% bupivacaine with NeoSTX increased intensity and duration of nocifensive blockade to thermal stimulus without increasing neurotoxicity or lethality. When bupivacaine is added to 3 mcg/kg NeoSTX, a weight-scaled dose approximately twice that previously used in humans, the percentage of animals effectively blocked increases from 10 to 100%. At clinically relevant doses (1 and 2 mcg/kg) the combination of NeoSTX with bupivacaine led to a significant increase in the intensity and duration of sensory-nocifensive blockade.

[000103] In Figures 4A-C, the intensity and duration of sensory blockade in groups of rats receiving either Bupivacaine 0.2% plain (Figure 4A), or Bupivacaine 0.2% with NeoSTX 3 mcg/kg (Figure 4B), or 0.2% Bupivacaine with NeoSTX 3 mcg/kg and Epinephrine 5 mcg/ml (Figure 4C). It is evident that the three-way combination gives more intense and prolonged blockade compared to the two-way combination. This predicts that the three-way combination will provide more intense and prolonged pain relief than the two-way combination, which is particularly important in the "high volume" and "medium volume" clinical scenarios as described above.

[000104] A model was used to study systemic toxicity. An intramuscular (ischial perineural) injection model was used to simulate systemic toxicity at an extravascular site, as intended for nerve block or infiltration in clinical use. In this model, co-injection of bupivacaine with NeoSTX slightly reduces systemic toxicity compared to animals receiving the same dose of NeoSTX in saline. In this model, the dose of bupivacaine is below the range that produces cardiotoxicity. The presumed mode of death is respiratory muscle weakness and some vasodilation due to NeoSTX, and bupivacaine may reduce systemic toxicity of NeoSTX by local vasoconstriction at the site of application, thereby delaying systemic absorption of NeoSTX. This conjecture is based on the known local vasoconstrictor effects of bupivacaine and previous experiments on regional blood flow in rats following ischial perineural injection of another site 1 sodium channel blocker, tetrodotoxin.

[000105] Since co-injection of bupivacaine increases the potency and duration of the NeoSTX nerve block while slightly reducing its systemic toxicity, the combination of NeoSTX-bupivacaine provides a significant improvement in the therapeutic index compared to NeoSTX in isolation. A formulation of NeoSTX developed for clinical trials provides a longer lasting sciatic nerve block than bupivacaine in rats. Co-injection with bupivacaine improves block reliability and duration compared to NeoSTX alone.

Example 2: Phase 1 Clinical Study in Adult Humans

[000106] Researchers at the University of Chile, Santiago, and a small biotechnology company, Proteus S.A, have developed a bioreactor process to efficiently produce clinical grade NeoSTX with excellent purity, stability, sterility and non-pyrogenicity. A series of preclinical toxicological safety studies in mice, rats, and sheep were performed at CHB and Toxikon, Inc. With approval oversight from the regional IRB and the Chilean Instituto de Salud Publica (ISP), the lead regulatory agency for drug approval and oversight in Chile, Dr. Rodriguez-Navarro and colleagues conducted two voluntary Phase 1 trials (Rodriguez-Navarro. et al., Anesthesiology, 2009, 106: 339-345; Rodriguez-Navarro, et al., Neurotox. Res., 2009; 16: 408- 15) and two Phase 2 trials of NeoSTX as a long-lasting local anesthetic for patients undergoing laparoscopic cholecystectomy ( Rodriguez-Navarro, et al., Reg. Anesth. Pain Med., 2011, 36:103-109; Rodriguez- Navarro - abstract for presentation at ESRA meeting, Sept. 2010).

[000107] In voluntary Phase 1 trials, doses of 10 or 50 mcg produced skin numbness with an excellent level of local and systemic safety. Combinations of NeoSTX with either bupivacaine or epinephrine dramatically enhances and prolongs the duration of skin numbness compared to NeoSTX alone, which in turn gave more prolonged skin numbness than Bupivacaine (Rodriguez-Navarro, et al., Neurotox. Res., 2009; 16: 408-15). Addition of epinephrine reduces systemic toxicity, and produces robust prolongation of sensory block.

[000108] In Phase 2 double-blind superiority trials of patients undergoing laparoscopic cholecystectomy under general anesthesia, comparing 100 mcg NeoSTX to bupivacaine for infiltration of entry sites, NeoSTX provided more prolonged analgesia than bupivacaine, and global ratings of self-reports indicated faster postoperative recovery. To date, 181 patients in Chile have received NeoSTX in clinical trials without any serious adverse events.

[000109] Recent human studies have demonstrated the potency, safety, and prolonged analgesic effects of NeoSTX using 10 mcg or 50 mcg skin infiltration in Phase 1 studies with awake volunteers, and using wound layer infiltration with 100 mcg in Phase 2 studies with patients undergoing laparoscopic cholecystectomy under general endotracheal anesthesia.

[000110] In awake volunteers, there were no reports of weakness and there were no signs of respiratory or hemodynamic abnormalities, but detailed surrogate measurements of subclinical systemic effects were not performed.

[000111] In patients in the laparoscopic cholecystectomy trials, with double-blind comparison to bupivacaine, injections were performed under general endotracheal anesthesia with controlled ventilation. Therefore, immediate effects on symptoms and respiratory muscle strength could not be assessed. However, all subjects had excellent respiratory effort at the end of surgery and none of the patients had a delay in extubation (surgery duration was approximately 1 hour), and no signs of weakness or hypoventilation were observed on prolonged observation in the Care Unit. Post Anesthetics (Post Anesthesia Care Unit - PACU).

[000112] However, although these Phase 2 Studies in surgical patients suggest good systemic safety conditions for NeoSTX at doses of 100 mcg in anesthetized subjects under mechanical ventilation, it was essential to perform dose scaling studies from additional Phase 1 Studies starting with lower doses and using alternative, more accurate measurements of subclinical systemic effects to select a safe dose (with a minimal adverse event threshold) in awake male young adult subjects.

Materials and methods

[000113] The main objective of this Phase 1 Study was to evaluate the systemic safety of neosaxitoxin (NeoSTX), given by subcutaneous injection in combination with the commonly used local anesthetic, bupivacaine, and Epinephrine. The secondary objectives of this Phase 1 Study were to establish the dose dependence of intensity and duration of cutaneous sensory block (numbness to thermal and mechanical stimuli) of NeoSTX in saline compared to NeoSTX in combination with bupivacaine and Epinephrine, to determine the safety and local tolerability of NeoSTX when given by subcutaneous injection, and to measure the pharmacokinetics of NeoSTX following subcutaneous injection. A Phase 1 Study was conducted under an investigator-initiated FDA IND to further establish the systemic and local dose and safety response to cutaneous sensory blockade of NeoSTX via subcutaneous infiltration in healthy young adult male volunteers.

[000114] As detailed below, tingling of the lips, tongue and fingers occurs at doses that appear to be physiologically very safe. This protocol change reflects an expectation that NeoSTX would be used in two dose ranges in subsequent Phase 2 Studies: one that produces minimal systemic symptoms in awake subjects and a second, slightly higher dose range that is intended for use in sedated or anesthetized in an operating room environment. This change involves administering cohorts of subjects in an operating room setting and allows subjects to receive low-dose anxiolysis with midazolam if the tingling is perceived as severe or very bothersome. Therefore, it is important to define safety in a dose range where the drug would be used in an operating room environment. A sequential dose escalation design will be used with groups of 9 subjects at each dose step. In a double-blind fashion, each participant received two injections simultaneously in a 3 cm x 3 cm square area on the skin of the posterior aspect of the leg (calf), one on each side, in a randomized block design. Each individual received an injection with 0.2% Bupivacaine alone on each side. On the other side, they received one of two possible solutions: (1) saline placebo, or (2) NeoSTX in combination with bupivacaine and epinephrine. In each dose group, only one of the injections involves a placebo.

[000115] Test subjects were young adult males. Subcutaneous infiltration was performed using a 25 gauge, 1% inch needle. The 10 ml volume was distributed, so that 2 ml is given subcutaneously on each side of the 3 x 3 cm square, and 2 ml is given across the diagonal.

[000116] Subjects were given NeoSTX at a dose of 5, 10, 15, 20, 30, or 40 mcg, 0.2% bupivacaine, and 5 mcg epinephrine/ml. Epinephrine was routinely added to local anesthetic injections for two reasons: (1) to delay systemic absorption and reduce systemic blood concentrations and thereby reduce the risks of systemic toxicity, and (2) to prolong the duration of local anesthesia. .

[000117] The use of an intra-subject combined dosing design with bupivacaine on one side and either NeoSTX in saline or NeoSTX in bupivacaine on the other allows intra-subject comparison of the intensity and duration of cutaneous analgesia with increasing doses of NeoSTX, with or without a fixed concentration of Bupivacaine 0.2%, compared to the currently used standard, Bupivacaine 0.2%.

[000118] The primary outcome measure of this Phase 1 study is the absence or presence of, the frequency, and the grade of AEs, as defined by 21 CFR 312.32 as a function of NeoSTX at each dose step, given with or without bupivacaine. These adverse events measure the systemic effects that are due to NeoSTX.

[000119] The project is intended to set a threshold for systemic effects, reducing the likelihood of adverse events and Serious Adverse Events.

[000120] An adverse event (AE) refers to any undesirable medical occurrence associated with the use of neosaxitoxin in humans, regardless of whether they are considered drug-related, as described in 21 CFR 312.32. The intensity (grade) of each AE was rated using the following scale: Grade 1 (Mild): Experiences that are generally transient, do not require special treatment, and do not interfere with the individual's daily activities. Grade 2 (Moderate): Experiences that introduce a certain level of inconvenience or concern to the subject, and may somewhat interfere with daily activities, but are usually alleviated by simple therapeutic measures (may include drug therapy). Grade 3 (Severe): Experiences that are unacceptable or intolerable, significantly disrupt the subject's usual daily activity, and require systemic drug therapy or other treatment. Grade 4 (Life-threatening): Experiences that place the subject in imminent danger of death. Grade 5 (Death): Subject fatality Results for NeoSTX in combination with bupivacaine and Epinephrine

[000121] Full evaluation of dose cohorts for the 5 mcg, 10mcg, 15mcg, 20mcg, and 30 mcg level has been completed and the 40mcg cohort (n=10, still ongoing) is nearing completion. At the 40mcg dose, a high rate of systemic effects in awake subjects was observed. As before, these effects involve systemic symptoms (tingling and numbness, and now nausea), but no signs of imminent physiological risk. The presence of nausea and vomiting for a short period of time (one hour) after the injection was also observed in 8/10 patients. To date patients have rated this as slightly boring. There was no impairment of oxygenation or ventilation, no impairment of inspiratory force or vital capacity (respiratory muscle strength) and no impairment of grip strength, no changes in heart rhythm, and no hypotension.

[000122] In future clinical use for infiltration, it is likely that higher injection volumes, eg 30 mls, will be used. In principle, blocking a surgical field that is three times as large requires approximately three times the injected volume of local anesthetic, and therefore three times the dose of local anesthetic, when given at a fixed concentration. Systemic safety varies with total dose, whereas local efficacy and duration vary with local drug concentrations. The study showed that 30 mcg NeoSTX is a safe dose in these volunteers.

[000123] At a total dose of 10 mcg in the injected material of 10 ml, the local concentration of NeoSTX equals the expected concentration when used for wound infiltration in a volume of 30 ml.

[000124] High dose of NeoSTX group (30 mcg) originates the maximum total dose for the proposed bottles of 30 ml future infiltration analgesia.

[000125] Figures 5A and 5B show the degree of sensory block. The Y-axis shows mechanical detection thresholds using Von Frey filaments. Von Frey numbers are logarithmically related to grams of applied force. Numbers > 16 represent fairly dense sensory block. Numbers 10-14 represent partial block and probably still contribute to degrees of postoperative analgesia. X axis is time after injection. Error bars show SD.

[000126] Figure 6 is a graph of the percentage of subjects having systemic symptoms: tingling, numbness, dizziness, nausea or vomiting at any point in time after administration of 0, 5, 10, 15, 20, 30 or 40 mcg of NeoSTX-bupivacaine-epinephrine. Nausea was observed in 80% of subjects at 40 mcg NeoSTX. No individual required medical intervention or supplemental oxygen. 02 sat, BP, NIF, VC, grip strength, ECG all very encouraging. For NeoSTX-Saline and NeoSTX-bupivacaine, transient mild tingling of the lips, tongue, and fingers began at 10 mcg, with tingling increasing with worsening at 40 mcg. Transient nausea and vomiting at 40 mcg. Symptoms usually resolved within 1 hour. There were no local or systemic sequelae observed.

[000127] Figures 7A and 7B are graphs of measurement of partial and dense blockade threshold, mechanical (Figure 7A) and cooling detection (Figure 7B) for NeoSTX, NeoSTX + bupivacaine, NeoSTX + bupivacaine + epinephrine, compared to placebo and controls (in NeoSTX), over time in hours.

[000128] NeoSTX-Bupivacaine produces longer mechanical and thermal block than plain NeoSTX or plain bupivacaine. NeoSTX-Bupivacaine gives reliable surgical anesthesia for 12 hours, strong reliable analgesia for >24 hours.

[000129] Based on partial mechanical block recovery, we can predict that if these formulations are used for peripheral nerve blocks, motor block will not persist >24 hours, which is desirable. Based on partial mechanical block recovery prior to thermal sensing partial block recovery (which correlates with pain sensation), an extended period of analgesia in the range of 24 - 48 hours, or even longer, with touch recovery Partial and motor function within 24 hours is predicted.

[000130] NeoSTX-Bupivacaine-Epinephrine produces longer mechanical and thermal blockade than NeoSTX-bupivacaine, straight NeoSTX, or straight bupivacaine. NeoSTX-Bupivacaine gives reliable surgical anesthesia/very dense analgesia for at least 24 hours. Based on the duration of the partial thermal detection block (which correlates with the sensation of pain), a prolonged period of postoperative analgesia in the range of 48-72 hours, or even longer, is predicted.

Claims (25)

1. Dosage unit for the treatment or prevention of pain in an awake, sedated or anesthetized human being characterized by the fact that it comprises an effective amount of - between 5 and 40 mcg of neosaxitoxin (NeoSTX); - a local anesthetic selected from the group consisting of bupivacaine, levobupivacaine, tetracaine and ropivacaine in a concentration range between 0.1% (1 mg/ml) and 0.5% (5 mg/ml); and - epinephrine in a concentration range between 2 mcg/ml (1:500,000 in common terminology) and 10 mcg/ml (1:100,000) to produce a minimum effective concentration at the administration site between 0.1 - 1 mcg NeoSTX/ ml). 2. Dosage unit, according to claim 1, characterized by the fact that the human being is awake. 3. Dosing unit, for high volume use of 35 - 120 ml for adult humans and 0.5 - 1.8 ml/kg for children, characterized by the fact that it comprises - Bupivacaine in a concentration range between 0.1 % (1 mg/ml) and 0.25% (2.5 mg/ml), giving a total systemic dose of bupivacaine of no more than 225 mg in adults or 2.5 mg/kg in children; - NeoSTX in a concentration range from 0.1 mcg/ml - 1 mcg/ml, giving a total systemic dose of 3.5 - 100 mcg in adults or 0.05 - 1.5 mcg/kg in children; and - Epinephrine in a concentration range between 2 mcg/ml (1:500,000 in common terminology) and 10 mcg/ml (1:100,000). 4. Dosage unit according to claim 3, characterized in that it is in an amount effective for infiltrating multiple layers of a large surgical wound for a complete open laparotomy, thoracoabdominal incision, or flank incision. 5. Dosage unit, according to claim 3, characterized by being in an effective amount for cesarean delivery, open hysterectomy, esophago-gastrectomy, nephrectomy, or major abdominal cancer surgeries. 6. Dosage unit according to claim 3, characterized in that it is in an effective amount for infiltration of the wound for total hip replacement or total knee replacement. 7. Dosing unit for medium volume use from 15 to 50 ml, characterized in that it contains - Bupivacaine in a concentration range of 0.125% - 0.3% (1.25 - 3 mg/ml), giving one dose systemic in adults of not more than 150 mg and not more than 2 mg/kg in children, - NeoSTX in a concentration range from 0.2 - 2 mcg/ml, giving a systemic dose in adults of 7 - 100 mcg and 0.1 - 1.5 mcg/kg in children, and - Epinephrine in a concentration range from 2 mcg/ml (1:500,000 in common terminology) and 10 mcg/ml (1:100,000). 8. Dosage unit according to claim 7, characterized in that it is in an effective amount for peripheral nerve blocks or plexus blocks. 9. Dosing unit, according to claim 7, characterized in that it is in an effective amount for infiltration. 10. Dosage unit, according to claim 7, characterized in that it is in an effective amount for shoulder, hand or arm surgery, infiltration or ilio-inguinal/ilio-hypogastric blocks for inguinal hernia correction, penis block for hypospadias correction, femoral block for total knee replacement or anterior cruciate ligament repair, intercostal nerve blocks for open chest surgery, or femoral and sciatic nerve blocks for leg amputation or foot and ankle surgery. 11. Dosage unit according to claim 10, characterized in that it is in an effective amount for nerve block, lumbar plexus block or lower volume sciatic block for the waist or knee for joint replacement surgery. 12. Dosage unit, according to claim 7, characterized in that it is for medium volume uses, in effective amounts to provide anesthesia for surgery, for periods of between 3 and 12 hours, analgesia after surgery, for at least 24 hours and recovery from a motor block to allow some strength in limb movement within 24 to 48 hours after administration. 13. Dosage unit for treating or preventing pain in an awake human being, consisting of (a) an effective amount of between 5 and 40 mcg of neosaxitosin in a volume between about 5 ml and about 120 ml, inclusive, and a concentration between 0.1 and 1 mcg of neosaxitoxin/ml; (b) a local anesthetic selected from the group consisting of bupivacaine, levobupivacaine and ropivacaine; and (c) optionally, one or more excipients. 14. Dosage unit, according to claim 13, characterized by being in an effective amount for cesarean delivery, open hysterectomy, esophagus-gastrectomy, nephrectomy, or major abdominal cancer surgeries. 15. Dosage unit according to claim 13, characterized in that it is in an effective amount for wound infiltration for total hip replacement or total knee replacement. 16. Dosage unit, according to claim 13, for the use of an average volume of 15 to 50 ml, characterized in that it comprises - Bupivacaine in a concentration range of 1.25-3 mg/ml, giving a systemic dose in adults not more than 150 mg or not more than 2 mg/kg in children; and - Neosaxitoxin in a concentration range from 0.2-1 mcg/ml. 17. Dosage unit according to claim 16, characterized in that it is an effective amount for peripheral nerve blocks. 18. Dosage unit according to claim 16, characterized in that it is in an effective amount for infiltration of a wound. 19. Dosage unit, according to claim 16, characterized in that it is in an effective amount for shoulder, hand or arm surgery, infiltration or ilio-inguinal/ilio-hypogastric blocks for inguinal hernia correction, penis block for hypospadias correction, femoral block for total knee replacement or anterior cruciate ligament repair, intercostal nerve blocks for open chest surgery, or femoral and sciatic nerve blocks for leg amputation or foot and ankle surgery. 20. Dosage unit according to claim 16, characterized in that it is in an effective amount for nerve blocks to the waist or knee for joint replacement surgery. 21. Dosage unit, according to claim 13, for medium volume uses, characterized in that it is in effective amounts to provide anesthesia for surgery for periods of between 3 and 12 hours, analgesia after surgery for at least 24 hours and recovery from a motor block to allow some strength in limb movement within 24 to 48 hours after administration. 22. Dosage unit, according to claim 13, for low-volume and long-term use, characterized in that it comprises bupivacaine at a concentration of 2.5-5 mg/ml, in which a dosage of 5-15 ml gives a systemic bupivacaine dose in adults of no more than 75 mg. 23. Dosage unit according to claim 22, characterized in that it is in an effective amount to provide lumbar sympathetic blockade for complex regional pain syndrome/reflex sympathetic dystrophy or leg vascular insufficiency or for celiac plexus blockade for pancreatitis or pancreatic cancer. 24. Dosage unit according to claim 22, characterized in that it is in an effective amount to provide a sciatic nerve block of prolonged duration, where rapid motor recovery is not an issue, such as for a lower leg amputation . 25. Dosage unit, according to claim 13, characterized in that it is in an effective amount for peripheral nerve blocks and plexus blockade with motor effects in the arms and legs, allowing recovery from a motor blockade within 24 to 48 hours after administration.

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