METHOD FOR TREATMENT OF PAIN

Field of the Invention

The field of the invention is the area of the treatment of pain in humans, particularly intractable pain. Specifically, the present invention provides for the usage of elcatonin in the formulation of medicaments to be administered via the intrathecal route.

Background of the Invention

Calcitonin has been shown to be effective for the treatment of pain in humans where pain is the result of conditions including, but not limited to, osteoporosis, Paget's disease, phantom limb pain, reflex sympathetic dystrophy, bone fractures, ankylosing spondylitis and bone metastases of malignancy [see, e.g., Gennari and Agnusdei (1988) Current Therapeutic Research 44 _. : 712-721] . Calcitonin has been used in therapeutic compositions for its hypocalcemic and hypophosphatemic effects. Calcitonin has also been used to increase bone mass.

Narcotics such as morphine have also been used for the treatment of chronic pain in humans, have the disadvantages of addiction and the development of tolerance to nontoxic doses of morphine.

There is a long felt need in the art for a pain treatment in those patients who cannot rely on conventional analgesic treatments. Such a pain treatment should advantageously not have significant negative side effects or toxicity; further, there is a need for a pain treatment which is not associated with the development of tolerance. The present invention provides an effective treatment for pain, particularly for patients suffering from chronic and intractable pain, for example, those patients suffering from intractable pain associated with terminal cancer.

Summary of the Invention

The present method provides an improved treatment for the treatment of pain in mammals, particularly the treatment of intractable pain in humans. In the present invention, elcatonin is provided by intrathecal administration to a human suffering from pain. The elcatonin is dissolved in a vehicle suitable for intrathecal injection, for example, sterile physiological saline. Preferably, the medicament further contains human serum albumin at a concentration from about 0.03 to 0.1% mg/mL. The dosage range is from about 0.2 to about 15 IU elcatonin per kg body weight, more preferably from about 1.6 to about 6.4 IU elcatonin per kg body weight. The dose to be administered is preferably formulated in a standard volume for intrathecal administration,

(preferably about 0.5 to about 2.0 mL for single intrathecal injection) . Elcatonin is administered via the intrathecal route, in the dosage disclosed herein, in single or multiple doses. For multiple doses, administration can be at intervals up to 24 to 48 hrs. Administration can be via bolus injections or using a continuous or pulsed delivery pump using the intrathecal route. The intrathecal administration of elcatonin provides strong and prolonged analgesic effect which is not associated with significant undesirable side effects, such as addiction, and there is no significant development of tolerance. The pain treatment of the present invention is particularly helpful in patients suffering from intractable and severe pain, such as that of terminal cancer patients.

Brief Description of the Drawings

Figure 1 illustrates that elcatonin administered via intrathecal route increase hot plate latencies in a dose related fashion. Hot plate latencies (sec) were measured one hour after elcatonin administration.

Figure 2 illustrates the increase in rat hot plate latency after intrathecal administration of elcatonin at doses from 0.2 to 3.6 IU/kg body weight (-+-0.2 IU/kg, -*-0.8 IU/kg, -"-1.6 IU/kg, -x-2.4 IU/kg, -♦-3.6 IU/kg -D-physiological saline control)

Figures 3A-3B provide a comparison of the increase in rat hot plate latency after intrathecal administration of elcatonin (a) at doses from 2.4 to 6.0 IU/kg and morphine (b) at doses from 37.5 to 125.0 mg/kg. The maximum latency is 40 seconds in these studies.

Figure 4 provides a comparison of the time courses and increase in hot plate latencies of low-dose elcatonin (2.4 IU/kg, -•-) , morphine (37.5 mg/kg -▼-) or physiological saline (-A-) administered via the intrathecal route. Figure 5 provides a comparison of the time courses and increase in hot plate latency of intermediate doses of elcatonin

(4.4 IU/kg, -▼-) , morphine (75.0 mg/kg, -■-) or physiological saline (-A-) administered via the intrathecal route. Maximum possible latency is 40 seconds in these experiments. Figure 6 provides a comparison of the time courses and increase in rat hot plate latency of high-dose for elcatonin (6.0 IU/kg, -▼-) , morphine (125 mg/kg, -•-) or physiological saline (-■-) (control) administered via the intrathecal route) .

Figure 7 illustrates increases in rat hot plate latency after 5 day multiple intrathecal administrations of elcatonin at a dose of 4.4 IU/kg body weight (-■-) or physiological saline (-0-) or sham injection (-♦-) . The vertical arrows indicate the times when rats were injected. Hot plate latency was measured at 0, 1, 3 and 24 hrs after injection on study days 1, 3 and 5. Figure 8 illustrates increases in rat hot plate latency after 10 single daily intrathecal administration of elcatonin at a dose of 4.4 U/kg (-■-) or saline vehicles (-0-) or sham injection (-0-) separated by 48 hr nontreatment intervals. The vertical arrows indicate the times when rats were injected. Hot plate latency was measured at 0, 1, 3 and 24 hrs after injection on study day 1 3, 5, 7, 9 and 11.

Figure 9 illustrates increases in rat hot plate latency after 5 day multiple intrathecal administrations of elcatonin (at a dose of 4.4 IU/kg) with HSA (0.1%) . Hot plate latency was measured at 0, 1, 3 and 24 hrs after injection on study day 1, 3 and 5.

Detailed Description of the Invention

Elcatonin, disclosed in U.S. Patent No. 4,086,221 (Shumpei Sakakibara et al., filed May 3, 1976) , has the following structure :

Elcatonin differs from eel calcitonin in that the cysteine residues at the N-terminal and the seventh amino acid positions are replaced with an aminosuberic acid bridge, which provides a carbon-carbon bridge in place of the disulfide bond between the cysteine residues in the natural calcitonin molecule. Other aminosuberic acid-bridged molecules are described in U.S. Patent No. 4,086,221, incorporated by reference herein. The amino acid sequences for eel, human, porcine, salmon and rat calcitonins are given n SEQ ID Nos: 1, 2, 3, 4 and 5, respectively. Replacement of the N-terminal and seventh position cysteines with an aminosuberic acid bridge in the calcitonins of SEQ ID Nos : 2-5 also yields relatively stable, analgesically effective peptide compositions. U.S. Patent No.4, 977, 139 (Yamada, et al . filed October 25, 1989) , incorporated by reference herein, describes aqueous elcatonin compositions with improved stability to light, heat and shaking.

U.S. Patent Nos. 4,977,139 (Yamada et al, filed October 25, 1989) , 5,118,667 (Adams et al . , filed May 3, 1991) and 4,758,550 (Cardineaux et al . , filed December 3, 1986) appear to disclose the use of calcitonin and elcatonin compositions in the treatment of various types of bone and/or pain disorders, but none appears to disclose the intrathecal administration of elcatonin for the treatment of intractable pain.

Miseria et al . (1989) Tumori (Italy) __ :183-184 appeared to disclose the epidural administration of salmon calcitonin for treatment of intractable pain, but the data did not appear to support its use as an analgesic.

As used herein, an analgesic effect means that the result is an insensibility to, or a decrease in the perception of pain or of a painful or injurious stimulus without loss of consciousness.

Analgesia and antinociceptive are used synonymously in the present application.

As used herein, intractable pain is that pain which does not respond to conventional analgesics, and/or that pain which persists upon development of tolerance to a narcotic analgesic such as morphine. Intractable pain can be associated with medical conditions, including, but not limited to, cancer, bone fractures, osteoporosis, post-amputation pain and certain other medical conditions.

The rat is a preferred animal model for testing compounds administered by the intrathecal route, and the rat analgesiometric hot plate test is an accepted model system for the potentially effective treatment of pain.

As disclosed herein (see Example 8, 9; Tables 1, 2) all doses of elcatonin used produced potent and prolonged antinociception, showing an onset within 15 min post-injection, reaching a peak at 30-60 min, and lasting at least 6 hours post-injection in the rat/hot plate analgasiometric model system. Since the doses of elcatonin used often produced maximum analgesia obtainable using the hot plate test, there were no apparent differences in hot plate latencies obtained following elcatonin ranging from 0.8-3.6 IU/kg. A slightly lower response was obtained at the lowest dose (0.2 IU/kg) used. Nevertheless, even intermediate and minimal antinociceptive responses could be obtained with lower doses of elcatonin in a dose-related manner.

TABLE 1

ANALGESIC ACTIVITY (HOT PLATE LATENCY) OF ELCATONIN FOLLOWING SINGLE INTRATHECAL ADMINISTRATION IN THE RAT

(Mean ± SEM)

Hot Plato Latency (sac)

Dos No. e of

Time After Administration (hr) lU/k Rat g 8

0 0.25 0.6 1 3 6 24

11 2 ±

0 21 11 7 ± 09 11 5 ± 06 122 ±08 11 8 ± 1 1 0 5 11 1 ± 0 7 11 5 ± 0 5

106 ±

02 11 130 ± 1 1 129 ± 07 12 7 ± 09 13 1 ± 07 12 1 ± 05 108 ± 07 08

08 13 98 i 08 15 1 ± 1 5* ^** 150 ± 1 5" 152 ± 1 3 ^* * 200 ± 28** 123 ± 1 0 11 7 ± 1 0

1 6 13 92 ± 05 164 ± 1 2" 160 ± 1 0" 164 ± 1 1" 15 1 ± 09" 136 ± 08" 109 ± 04*

24 13 90 ± 07 200 ± 1 5" 201 ± 1 9" 195 ± 1 8 ^** 21 8 ± 2 1" 126 ± 1 2* 103 ± 1 2

104 ±

36 12 194 ± 1 6" 21 4 ± 1 8** 23 5 ± 25 ^** 195 ± 1 0" 157 ± 1 3" 102 ± 1 0 09

*p __ 005 student's t-test "p ≤ 0 05 student's t-test

In the rat hot plate analgesiometric assay data described herein, the intrathecal administration of elcatonin gave unexpectedly improved antinociceptive results as compared with intravenous injection of elcatonin (see Example 7 herein) . As shown in Example 9 and Figures 6-8, intrathecal elcatonin also provided dramatically improved analgesia in the rat hot plate assay as compared to intrathecal morphine. Furthermore, in a comparison of the analgesic activity of morphine and elcatonin administered intrathecally, elcatonin appeared to have significantly greater analgesic activity in the rat hot plate test, and the analgesic effect of the elcatonin also appeared to be of greater duration than that of morphine (see Example 9 and Figures 3-6 herein) . The following examples are provided for illustrative purposes, and they are not intended to limit the scope of the invention as disclosed and claimed herein. All studies were carried out in accordance with FDA GLP regulations (21 C.F.R. 58). Any routine procedural modifications and deviations from the disclosed practice of the invention disclosed herein which are obvious to

the skilled artisan are within the scope of the instant invention.

EXAMPLES

Example 1: Experimental Animals The rat is one of the preferred species for testing compounds for analgesic activity by various US and International Regulatory Agencies. Historical information concerning Spraque-Dawley rats is available in the published literature. Adult male Spraque- Dawley rats were obtained from Sasco Inc. (Oregon, I) , and were allowed a 7 day acclimation period prior to use in experimentation. Rats were weighed 1 day after receipt and again after 7 days or before assignment to an experiment. Rats were assigned to experiments and treatments randomly. Any animal whose body weight differed significantly from the mean body weight of group, which showed abnormal changes in weight or which showed signs of a physical disorder was not used. Test rats were in the range of 320-420 g.

Example 2: Animal Husbandry

Animal husbandry was in accordance with the Guide for the Care and Use of Laboratory Animal Resources, National Research

Council, DHHS, Publication No. NIH 85-23, 1986, the United States Department of Agriculture guidelines and Animal Welfare Act. Rats were housed in environmentally controlled rooms maintained at a temperature of 18 to 26 °C, generally from 18-20 °C, a relative humidity of 40 to 70% and with greater than ten room air exchanges per hour. A 12 hr light/dark cycle per day was maintained. The animals were provided access to tap water and to food (Agway Rat Chow, Agway) both ad libi tum. The inventors were not aware of any contaminants in either food or water supply which could have adversely affected the experiments.

Waste material was removed from cages twice a week, or more often if necessary. Cages and feeders were sanitized every two weeks unless required more often.

Once catheterized, rats were housed individually in stainless steel cages to avoid catheter damage or destruction by cagemates.

Each rat was identified by a uniquely assigned number on its cage.

Example 3 : Preparation of Stock Solutions

Elcatonin ( [Des-Cys ¹ ,ASU ⁷ ] -Calcitonin, Eel; carbacalcitonin) was obtained from Bachem, Inc. (Torrance, CA) . It was stored at -20 °C, protected from light. Stock solutions of elcatonin (1 mg/mL in sterile isotonic saline, USP) for use in tests of analgesic activity after intrathecal injection were made fresh daily and kept on ice. Appropriate dilutions were made just prior to injection.

Sterile isotonic saline solution (0.9% w/v NaCl, USP grade) was obtained from Sigma Chemical Co. (St. Louis, MO) .

Human serum albumin was obtained from Sigma Chemical Co. (St. Louis, MO) . Stock solutions were 25% (wt/vol) . Morphine sulfate was obtained from Mallinckrodt, Inc. (St. Louis, MO) . Stock solutions of morphine for intrathecal injection were prepared daily (10 mg/mL in sterile isotonic saline) and held on ice. Appropriate dilutions were made just prior to injection.

Example 4: Neurosurgery/Catheterization for Intrathecal

Administration of Pharmaceuticals Intrathecal catheters were surgically implanted in the rats after application of anesthetic (Nembutal, 30 mg/kg, intraperitoneal) 5-7 days prior to the test procedure. Each catheter (polyethylene tubing, PE 10) was inserted via a slit in the atlanto-occipital membrane, and the catheter was threaded through the subarachnoid space 7.5 cm so that the tip was positioned at the rostral margin of the lumbar enlargement. Each catheter was anchored to the skull with cyanoacrylate adhesive, and the skull was closed with wound clips. The free end of each catheter was heat annealed; when compositions were to be injected via the intrathecal route each composition was introduced therein using a Hamilton needle inserted into the sealed catheter. The 5-7 day period after catheter implantation prior to testing allowed for full recovery from the surgical procedures.

Example 5: Assessment of Analgesic Activity

Pain sensitivity was assessed using a standard analgesiometric hot plate test (Woolfe and MacDonald (1944) J. Pharmacol. Exp. Ther. £K):300) . The hot plate response was determined by placing a rat on a 55°C copper hot plate enclosed in a plexiglass cylinder. The interval (measured in seconds) between placement on the hot plate and the response of either licking the hind paws or jumping off the hot plate was defined as the "hot plate latency." If an animal did not respond to the hot plate by licking the hind paws or jumping off the hot plate, it was removed after 40 sec to prevent tissue damage to the paws, and the hot plate latency value assigned was 40.

Because of the technical demands of these experiments, no more than 8-9 rats were tested on a given day. Sufficient animals were studied to obtain 10-11 evaluable rats per test condition. On the study day, animals were tested for baseline hot plate responses, and then received bolus injections of test analgesic or physiological saline (control) . Hot plate responses were assessed again at 15 min, 30 min, 1 hr, 3 hr, 6 hr and 24 hr after intrathecal injection of test compound or vehicle control.

Example 6: Doses Administered

All doses of elcatonin were administered as bolus injections. The following intrathecal doses of elcatonin have been tested in the single administration studies: 0.2, 0.8, 1.6, 2.4, 3.6, 4.4, 5.2 and 6.0 IU/kg or 0.03, 0.13, 0.25, 0.39, 0.58, 0.71, 0.84 and 0.97 μg/kg. The intravenous doses of elcatonin tested were 10.0, 30.0, 100.0 and 300.0 IU/kg or 1.61, 4.83, 16.1 and 48.3 μg/kg. One microgram of elcatonin corresponds to about 6.2 IU.

Example 7: Volumes Administered All intrathecal doses were administered in volumes of 37.5 μL/kg followed by a catheter flush of 10 μL/kg physiological saline (single administration studies) or in volumes of 10 μL followed by a catheter flush of 8 μL of physiological saline (multiple administration studies) . All intravenous doses of elcatonin were administered in a volume 0.5 mL/kg.

Example 8 : Dose Response for Intrathecal Elcatonin in the Rat Analgesiometric Assay In this study, a range of doses of elcatonin administered by the intrathecal route was tested in the analgesiometric assay to establish the minimum and peak effective doses inducing analgesia and to determine dose responsiveness. The doses tested were 0.2,

0.8, 1.6, 2.4, and 3.6 IU/kg. The desired dose was injected in a volume of 37.5 μL/kg followed by a catheter wash-out of 10 μL saline via the intrathecal route. Baseline (pre-drug) and responses at 15 in, 30 min, 1 hr, 3 hr, 6 hr and 24 hr after the drug or vehicle injection were determined. Eleven to thirteen animals were used for each dose of elcatonin; 21 control vehicle (saline) animals were used in this study.

A total of 113 adult male rats, of weights of 350 g to 400 g, were used. Intrathecal catheters were surgically implanted as described in Example 4. Three rats died after the surgical implantation of the catheters but prior to the administration of the test composition. A fourth rat died 24 hr after the intrathecal administration of 0.8 IU/kg elcatonin. This study demonstrated that elcatonin administered by the intrathecal route increased hot plate latencies in a dose-related fashion (See Table 1, Figure 1) . The onset of antinociception was within 15 min after injection, and a statistically significant increase in the latency period occurred between 0.25 to 3 hrs. Significant antinociception was also apparent at 6 hrs after injection at the three higher doses. In most tests, the animals' hot plate latencies returned to baseline by 24 hr post-injection (See Table 1, Figure 2) .

Data were analyzed for statistical significance, and a point estimate (mean and standard deviation) was used to analyze analgesic activity for each dose of the drug for a selected time period. Nonparametric statistics were calculated to address the level of significance of any difference between control and test groups. The lowest dose tested (0.2 IU/kg elcatonin) did not produce a statistically significant response, but doses of 0.8 and 1.6 IU/kg resulted in significant antinociception while doses of 2.4 and 3.6 IU/kg resulted in strong antinociception (Figure 2) . The

administration of saline alone did not alter the baseline response in the control population.

Example 9: Analgesic Activity of Intrathecal Elcatonin vs. Intrathecal Morphine This study compared the analgesic activities of morphine and elcatonin, each administered by the intrathecal route in the rat animal model.

Nine-six (96) rats, ranging in weight from 320 g to 420 g, were used in this study. Catheters were implanted and recovery was allowed for as described hereinabove. Elcatonin doses were 2.4, 3.6, 4.4, 5.2, and 6.0 IU/kg (0.39, 0.58, 0.71, 0.84 and 0.97 μg/kg, respectively) . Morphine sulfate was administered in doses of 37.5, 75.0 and 125.0 μg/kg. Drug or saline vehicle was administered using Hamilton needles inserted into the catheters with dose volumes of 37.5 μL/kg followed by a 10 μL saline catheter wash-outs. Baseline (pre-drug) and responses at 15 min, 30 min, 1 hr, 3 hr, 6 hr and 24 hr after the drug or vehicle injection were determined.

Both elcatonin (Figure 3A) and morphine (Figure 3B) increased hot plate latencies, indicating analgesic effects (Table 2) . All tested doses of elcatonin produced potent and prolonged antinociception, showing an onset within 15 min post-injection, reaching a peak at 30 - 60 min, and lasting at least 6 hours post-injection (Table 2, Figure 3A) . Since the doses of elcatonin used often produced the maximum analgesia detectable using the hot plate test, there were no apparent differences in hot plate latencies obtained following elcatonin doses ranging from 3.6 - 6.0 IU/kg. A slightly smaller response was obtained when the lowest dose of elcatonin (2.4 IU/kg or 0.39 μg/kg) was used. Nevertheless, the results of Example 8 indicate that intermediate and minimal antinociceptive responses could be obtained with lower doses of elcatonin in a dose-related manner.

Morphine increased hot plate latency in a dose-related fashion, and the onset of analgesia occurred within 15 min post-injection (Table 2, Figure 3A) . However, the duration of analgesia appeared to be shorter than for elcatonin, with reduced

potency apparent by 3 hours post-injection, and a return toward baseline hot plate latency scores by 6 hours post-injection, particularly for the lower doses. In addition, the peak levels of analgesia achieved in morphine-injected animals were generally lower than that achieved with elcatonin-injected animals at the doses used in this study (Table 2, Figure 3A,3B) .

To compare analgesic potencies of elcatonin and morphine, doses were converted to nmol/kg. Dose-responsiveness to both drugs was plotted at several time points in order to estimate equivalent doses (Table 2) . At 30 min post-injection 0.12 nmol/kg elcatonin (2.4 IU/kg or 0.39 μg/kg) produced a hot plate latency similar to 98.84 nmol/kg morphine sulfate (75.0 μg/kg) and 0.18 nmol/kg elcatonin (3.6 IU/kg or 0.58 μg/kg) is approximately equivalent to 164.73 nmol/kg morphine sulfate (125.0 μg/kg) . Similar potencies are seen at 1 hour post- injection (Table 2) . However, at 6 hours post-injection morphine antinociceptive activity was reduced compared to elcatonin, such that all tested doses of elcatonin (0.12 - 0.30 nmol/kg) result in more potent hot plate analgesia than all tested doses of morphine sulfate (49.42 - 164.73 nmol/kg) at this time point (Table 2) .

TABLE 2

COMPARATIVE ANALGESIC ACTIVITY (HOT PLATE LATENCY) OF

ELCATONIN AND MORPHINE FOLLOWING SINGLE INTRATHECAL

ADMINISTRATION IN THE RAT (MEAN ± SEM)

Dose Hot Plate Latency (sec.)

No. of

Group Time After Administration (hr.) Rat

IUkg s «/kg nmolkg

0 025 05 1 3 6 24

1251 1416 1432 1432 1316 1314 1285

Control

15 - - - ± ± ± ± ± ± ± (vehicle) 060 064 100 102 079 074 187

1354

1970 2390 2468 130 121 108

9 24 039 012 ± ± ± ± ± ±

± 175* 312 180" 07* 05 07 191

1292 2104 2603 2608 2314 2037 1579

10 36 058 018 ± ± ± ± ± ± ± 14 222" 308" 369" 305" 344 240

1368 2189 2603 2620 2111 2002 1178

Elcatonin

9 44 071 022 ± ± ± ± ± ± ± 110 312* 336" 237" 218* 326 175

1341 2789 3155 2582 2881 2297 1236

8 52 084 026 ± ± ± ± ± ± ± 092 310" 417" 379" 341" 260" 169

1138 2522 2891 2848 2660 2469 1117

9 60 097 030 ± ± ± ± ± ± ± 089 413" 340" 335" 387" 284" 180

1281 2208 2097 2181 ±1997 1402 1133

9 - 375 4942 ± ± ± ± ± ± ± 123 268" 256* 246" 205* 124 151

1376 2276 2186 1988 1742 1766 1260

Morphine 9 - 750 9884 ± ± ± ± ± ± ± 127 243" 225" 204* 193 171 119

1328 194 2636 2683 2169 1586 1349

9 - 1250 16473 ± ± 1 ± ± ± ± 135 16" 420" 304" 362* 329 163

<005 I c

__ 001 (T-test) J f<ompared to baseline

To compare duration of analgesic activity, the time course of antinociception was compared for the lowest doses of elcatonin and morphine (Figure 4) , intermediate doses of elcatonin and morphine (Figure 5) , and high doses of elcatonin and morphine (Figure 6) . It is apparent that elcatonin has a generally longer duration of analgesic activity than morphine, particularly

apparent at low and intermediate doses. Vehicle injections did not produce detectable changes in hot plate latencies.

Body weight changes were unremarkable, and no mortality was observed. No clinical or behavioral abnormalities were noted within a 24 hour observation period following intrathecal administration of elcatonin. Short-term tail (2) and hind limb (1) myoclonia occurred in two animals (3.6 and 5.2 IU/kg) . In these experiments, no significant morbidity or clinical and behavioral abnormalities were observed after the intrathecal administration of elcatonin.

In summary, these result indicate that elcatonin produces potent analgesia at approximately 1000-fold lower molar concentrations than morphine sulfate when administered via the intrathecal route and that the analgesia produced by elcatonin is sustained for prolonged periods, at least up to 6 hours post-injection, as compared to morphine sulfate administered intrathecally.

Example 10: Analgesic Activity of Intravenous Elcatonin

This study sought to establish effective doses for analgesia of elcatonin administered by the intravenous route and to determine dose-responsiveness .

A total of 24 rats, ranging in weight from 320 to 420 g, was used in this study. Four to eight rats were used for each dose level. Elcatonin was administered in a volume of 0.5 ml/mg body weight by tail vein injection. The doses used were 10, 30, 100 and 300 IU/kg (1.61, 4.83, 16.1 and 48.3 μg/kg, respectively) . Baseline hot plate responses and responses at 15 min, 30 min, 1 hr, 3 hr, 6 hr and 24 hr after injection were determined in the hot plate analgesiometric assay described in Example 5 hereinabove.

The results in this study indicated that elcatonin injected intravenously produced only small increases in hot plate latencies, reflective of only small analgesic effects (Table 3) .

TABLE 3

ANALGESIC ACTIVITY (HOT PLATE LATENCY) OF ELCATONIN FOLLOWING SINGLE INTRAVENOUS ADMINISTRATION IN THE RAT

(Mean ± SEM)

Dose No. Hot Plate Latency (sec.)

Group of lU/k Time After Administration (hr.) κg/kg Rat g 8 0 0.25 0.6 1 3 6 24

12.03 11.95 15.64 14.95 14.11 10.45 8.86

10.0 1.61 8 ± ± ± ± ± ± ± 1.02 1.40 2.39 2.92 0.94 1.03 1.23

10.71 10.04 12.18 14 14* 14.26 15.12 10.76

30.0 4.83 8 ± ± ± ± ± ±

Elcatoni ± 0.96 1.22 1.14 1.39 2.20 1.25 n 1.02

100. 13.53 11.05 9.63 11.20 14.76 14.00 14.30

16.1 4 0 ± ± ± ± ± ± ± 1.63 1.99 1.21 1.95 1.19 1.83 1.33

11.22 11.05 11.65 12.65 17.37 11.55 10.52

300.

48.3 4 0 ± ± ± ± ± ± ± 0.38 3.82 2.31 2.09 4.25 2.80 1.74

No increases in apparent analgesic effects were observed for the higher doses (100 and 300 IU/kg) as compared with the lower dose ranges (10 and 30 IU/kg) although the peak effects appeared to occur late (at 3-6 hr) and slightly prolonged for the 30-300 IU/kg doses as compared to 30 min for the 10 IU/kg dose. No significant dose response effect was observed for elcatonin administered by the intravenous route at doses from 10 to 300 IU/kg body weight in the rat (Table 3) .

A comparative analysis of the hot plate latencies obtained after intravenous and intrathecal administration of elcatonin indicates that at 0.5 hr the lowest intravenous dose of elcatonin (10.0 IU/kg or 1.61 μg/kg) produces a hot plate latency similar to that produced by 1.6 IU/kg (0.25 μg/kg) of elcatonin administered via the intrathecal route (See Example 8) .

In these experiments, no significant body weight changes, morbidity, mortality or clinical and behavioral abnormalities were observed after the intravenous injection of elcatonin.

Example 11: Analgesic Activity of Multiple Intrathecal Doses of

Elcatonin This study assesses the effectiveness of repeated doses of intrathecally administered elcatonin for analgesia. Intrathecal

catheters were implanted in rats as described hereinabove, and a 7-10 day period thereafter allowed for full recovery from the surgical procedures.

Eighteen rats were assigned to three treatment groups of six rats per group. The study is conducted in two treatment phases at one week intervals. Treatment Group I rats receive no treatment. Group II rats receive five consecutive daily intrathecal doses of sterile physiological saline (vehicle control) . Group III rats receive five consecutive daily doses of elcatonin (4.4 IU/kg body weight) .

For each treatment, hot plate latencies are determined as described in Example 5 hereinabove at 0 minutes (predose, baseline) , 1 hour, 3 hours, and 24 hours following injection on the first, third, and fifth study days. The results in Figure 7 show that hot plate latency is increased by repeated intrathecal doses of elcatonin, and it appears that at 24 hr after each successive injection, there is greater hot plate latency, and a cumulative analgesic effect over time. In these experiments, no significant body weight changes, morbidity, mortality or clinical and behavioral abnormalities were observed after the intrathecal administration of elcatonin.

Example 12 : Analgesic Activity of 10 Single Daily Intrathecal Doses of Elcatonin Over 11 Days in the Rat This study assesses the effectiveness of repeated doses of 10 single consecutive daily doses of elcatonin administered via the intrathecal route in the rat during two 5 day treatment phases separated by 48 hours nontreatment interval.

Intrathecal catheters were implanted in rats as described hereinabove, and a 7-10 day period thereafter allowed for full recovery from the surgical procedures. Nine rats were assigned to three treatment groups of three rats per group. The study is conducted in two 5 day treatment phases separated by 48 hours nontreatment interval. Treatment Group I rats receive no treatment (sham injection) . In each treatment phase, Group II rats received five consecutive daily intrathecal doses of sterile physiological saline (vehicle control) , for a total of 10 injections per study treatment. In each treatment phase, Group

III rats each received five consecutive daily doses of elcatonin (4.4 IU/kg body weight) , total 10 injections per study treatment (Table 4) .

For each treatment, hot plate latencies are determined as described in Example 5 herein above at 0 minutes (predose, baseline) , 1 hour, 3 hours, and 24 hours following injection on the 1st, 3rd, 5th, 7th, 9th and 11th study days (Table 4) .

The results in Figure 8 show that hot plate latency is increased by repeated intrathecal doses of elcatonin, and there is a cumulative analgesic effect over time.

TABLE4 STUDYDESIGN

Study Day

Procedure

1 | 2 | 3 | 4 | 5 . 1 7 8 | 9 10 11 12

Phase 1

Injection X X X X X

Hot plate X X X test

Metabolic X X X X X X observation

Clinical X X X X X X observation

Phase 2

Injection X X X X X

Hot plate X X X test

Metabolic X X X X X X observation

Clinical X X X X X X observation

In these experiments, no significant body weight changes, morbidity, mortality or clinical and behavioral abnormalities were observed after the intrathecal of elcatonin.

Example 13 Human Serum Albumin-Containing Pharmaceutical Compositions This study is conducted to evaluate the effect of human serum albumin as an ingredient in elcatonin-containing analgesic

compositions. Human serum albumin (HSA) is a candidate to prevent adherence of elcatonin to the vials or tubing or the like before administration to a patient or experimental animal.

On each study day, a standard 25% (wt/vol) solution of HSA is diluted under aseptic conditions with physiological saline (0.3% solution of NaCl, USP) to achieve the desired stock concentration of 0.2% (wt/vol) . Where needed, an aliquot of elcatonin solution is mixed with an equal volume of 0.2% HSA to give a final concentration of 0.1% HSA in the elcatonin-containing composition to be administered.

Thirty-two male rats (320-420 g) are obtained, adapted, catheterized and recovered as described hereinabove. Group I rats are injected intrathecally with elcatonin in physiological saline at a dose of 4.4 IU/k in a volume of 10 μl/kg. Group II rats are injected intrathecally with elcatonin in physiological saline containing 0.1% HSA at a dose of 4.4 IU/kg, in a volume of 10 μL/kg. Group III rats are injected with physiological saline containing 0.1% HSA, 10 μL/kg. Each dose is followed by a catheter washout of 8 μL of physiological saline. In each of the study weeks, hot plate latencies are determined at 0 minutes (baseline, predose) , 1 hour, 3 hours, 6 hours, and 24 hours following intrathecal injections on the 1st, 3rd and 5th study days . The results in Figure 9 show increased rat hot plate latency after 5 day multiple intrathecal administrations of elcatonin (at a dose of 4.4 IU/kg) in HSA (0.1%) .

In these experiments, no significant body weight changes, morbidity, mortality or clinical and behavioral abnormalities were observed after the intrathecal administration of elcatonin.

Example 14 : Pharmacokinetics of ¹⁵ I-Elcatonin in the Rat Following Intravenous and Intrathecal Administration

Pharmacokinetics/mass balance and tissue distribution in selected tissues were studied in 21 male Sprague-Dawley rats according to the study design described in Table 5.

Radioiodinated elcatonin ( ¹²⁵ I-Elcatonin) was administered intravenously (IV) via jugular vein and intrathecally (IT) via a surgically implanted catheter. Blood, plasma, urine, feces, tissue and carcass samples were collected at specified times

(Table 5) following a single 1 μg/kg IT bolus dose or a single 50 μg/kg IV bolus dose.

TABLE5

STUDY DESIGN AND SCHEDULE OF SAMPLE COLLECTION

Anlma

Dosln

1 Biological Samples for Total

Dose(s) Sampling Times

Group g Radioactivity Measurement

Route

(n)

8 plasma samples per animal

Single dose Blood

Brain, spinal cord, lung, liver,

5 mm, 0 5. 1, 2, 4, 8, 24 and thyroid, kidney, blood and

1 IV 50 pg/kg of ""l-Eteatomn 48 hr post-dose plasma samples from 2

(n=4) (Bolus) ( ₂ 0 pci/pg) animals

Tissues, blood and plasma

Injection volume 1 mL/kg 120 hr post-dose (n=2 rats)

(A total of 34 plasma, 2 blood, and 12 tissue samples)

8 plasma samples per animal

Single dose Blood

Brain, spinal cord, lung, liver,

0 5. 1. 2, 4, 6, 8, 24 and 48 hr thyroid, kidney, blood and

2 Intra¬ 1 pg kg of '"l-Elcatonin post-dose plasma samples from 2 (n=4) thecal (20 pci/pg) animals

Tissues, blood and plasma

Injection volume 20 pL kg 120 hr post-dose (n=2 rats)

(A total of 34 plasma, 2 blood, and 12 tissue samples)

Unne

7 unne and 5 fecal samples per

0-β, 6-12, 12-24, 2 - 8, ₄ 8-72, animal

Single dose 72-96 and 96-120 hr post-dose

1 Carcass sample per animal

3 IV 50 pg kg of ^ι:B l-Elcatonιn Feces

(n=4) (Bolus) (20 pci/pg) 0-2 ₄ , 2 -48, 48-72, 72-96 and 96-

(A total of 4 carcass, 28 unne 120 hr post-dose and 20 fecal samples)

Injection volume 1 mL kg

Carcass

Cage washes

120 hr post-dose

Brain, spinal cord, lung, liver

Single dose thyroid, kidney, blood and plasma samples per each Tissues, blood and plasma

4 IV 50 pg/kg of ^,a l-Elcatonιn animal 1 hr post-dose (n=2 rats) and

(n=4) (Bolus) ( ₂ 0 pci/pg) 24 hr post-dose (n=2 rats)

(A total of 24 tissue, 4 blood

Injection Volume 1 mL kg and 4 plasma samples)

Brain, spinal cord, lung, liver,

Single dose thyroid, kidney, blood and plasma samples per each Tissues, blood and plasma

5 Intra¬ 1 pg/kg of ^,2S l-Elcatonιn animal 1 hr post-dose (n=2 rats) and (n=4) thecal (20 pci/pg) 24 hr post-dose (n=2 rats)

(A total of 24 tissue, 4 blood

Injection Volume ₂ 0 pUkg and 4 plasma samples)

Urine, feces, blood plasma,

6 non- Samples were collected at 24

No dose tissues, carcass and cage

(n=1) dosed hours for analysis of background washes

Total radioactivity was measured using a Gamma Counter. In addition, protein-associated radioactivity in plasma was

estimated by measuring radioactivity in the plasma proteins after precipitation with trichloroacetic acid.

The terminal phase half-life of total radioactivity (free I ₂ and iodinated proteins) in plasma was 29 h following intravenous dosing and 32 h following intrathecal dosing. The terminal phase half-lives for TCA-precipitable radioactivity following intravenous and intrathecal dosing were similar (estimated to be 40 h) .

Following intrathecal dosing, drug-derived radioactivity distributed relatively rapidly to tissues (C _max at 1 h) . The rank order of concentration was thyroid>spina1 cord>brain>kidney>blood>lungs>liver. Without wishing to be bound by any particular theory, the high concentration of radioactivity in the thyroid is most likely a consequence of the presence of free I ₂ in the dosing preparation. The brain plasma radioactivity levels were significantly higher after intrathecal administration than after intravenous dosing, indicating poor crossing of the blood/brain barrier. Eighty-six percent of the dose was recovered after IV dosing. Approximately 46% of the administered radioactivity was recovered in the first 24 hrs. The major route of elimination was via the urine (71% dose) .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Noble, John F.

Abajian, Henry B. (ii) TITLE OF INVENTION: Method of Treatment of Pain

(iii) NUMBER OF SEQUENCES: 5

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Greenlee, Winner and Sullivan, P.C. (B) STREET: 5370 Manhattan Circle, Suite 201

(C) CITY: Boulder

(D) STATE: Colorado

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(F) ZIP: 80303 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO

(B) FILING DATE: 18-APR-1996

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/424,866

(B) FILING DATE: 18-APR-1995

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Ferber, Donna M.

(B) REGISTRATION NUMBER: 33,878 (C) REFERENCE/DOCKET NUMBER: 1-95A WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (303) 499-8080

(B) TELEFAX: (303) 499-8089

(2) INFORMATION FOR SEQ ID NO:1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : not relevant

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Eel

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gin Glu Leu

1 5 10 15

His Lys Leu Gin Thr Tyr Pro Arg Thr Asp Val Gly Ala Gly Thr Pro 20 25 30

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: not relevant

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Cys Gly Asn Leu Ser Thr Cys Val Met Leu Gly Thr Tyr Thr Gin Asp 1 5 10 15

Phe Asn Lys Phe His Thr Phe Pro Gin Thr Ala lie Gly Val Gly Ala 20 25 30

Pro

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANIS : Porcine

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Cys Ser Asn Leu Ser Thr Cys Val Leu Ser Ala Tyr Trp Arg Asn Leu 1 5 10 15

Asn Asn Phe His Arg Phe Ser Gly Met Gly Phe Gly Pro Glu Thr Pro 20 25 30

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Salmon

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gin Glu Leu 1 5 10 15

His Lys Leu Gin Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 20 25 30

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: not relevant

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (A) ORGANISM: Rat

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gin Asp Leu 1 5 10 15

Asn Lys Phe His Thr Phe Pro Gin Thr Ser lie Gly Val Gly Ala Pro 20 25 30