Implant preparations for delivery of bioactive macromolecules

This invention relates to bio-absorbable implant preparations containing bioactive macromolecule, which are capable of sustained action when implanted.

Drug administration by the oral route is applicable mostly to low molecular weight (∼850 daltons) compounds which are stable at acidic pH of the stomach and alkaline condition of the gastrointestinal tract. Bioactive macromolecules, such as heparin (Mol. Wt. 20,000 daltons), insulin (Mol. Wt. 6,000 daltons), somatotropin (Mol. Wt. 22,000 daltons), or the like are inactivated by proteolytic enzymes in the digestive tract, if taken by mouth. As a result, they can be administered only by injection when required. But injection usually introduces too high an initial dose which is absorbed in part as well as degraded by serous enzymes at the subcutaneous injection site resulting in rapid decay to an inadequately low level a few hours later. To compensate for the decay, a second injection is required. Another remedy to correct the inconsistency is to infuse a dilute solution of the labile agent continuously at a low rate. The slow infusion can actually achieve a better outcome, because most active agents have a relatively short half-life in vivo or are toxic if the daily required dose is given at once by injection. However, the advantage of low-dose infusion is compromised by the incidence of infection and discomfort due to the presence of the indwelling needle and the catheter attachment. Therefore, extensive effort is continuing to find an implantable infusion device or preparation that can deliver an active agent for a prolonged period of time.

In the early 1970's when the merit of giving a drug in small doses by continuous external infusion was demonstrated, further efforts were aimed at the development of implantable pumps and drug releasing capsules. Another purpose was to free the recipient of the chance of infection and provide total unrestrained mobility which often contributed immensely to the patient's sense of well being.

There have been several different designs of implantable pump under development during the past five years. But none has been long lasting without developing technical problems in a short time under stringent clinical conditions.

In a different approach, many existing and specially synthesized polymers have been evaluated for suitability as excipient for drug delivery implants. The advantages of such an implant are its relatively small size as compared to the pumps aforementioned, and the fact that the active component can be compounded with the excipient material directly for delivery which often lasts for months or even years. Recent efforts have been directed at the development of excipients that can be eroded and gradually absorbed in the body (J. Heller in Recent Advances in Drug Releasing Systems, Anderson &amp; Kim (eds) Plenum Press, 1984, N.Y., p.101). The erosion will also free a bioactive macromolecule incorporated therein, and make the removal later unnecessary. Several synthetic polymers are known to degrade to some extent and become absorbed once implanted in the body. However, current search has concentrated almost exclusively on the more promising polyesters as the excipient material. But, the novel polyesters often erode too slowly in the aqueous environment to be useful for delivery of bioactive macromolecules. Therefore, very few such experimental implants made therefrom have advanced even to the level of testing in laboratory animals. At present, there is only one procedure producing a polymer implant for polypeptide hormone such as insulin which has been clearly demonstrated to be capable of promoting growth and reducing hyperglycemia for many weeks (Langer et al., Diabetes, <u style="single">29</u> (1980) 37). But the device is non-absorbable in the body, and the long-term biological effect of the polymer material on the host still requires extensive evaluation.

US-A-4 452 775 describes delivery systems for releasing macromolecular active agents to a body site at a controlled rate for a prolonged period of time, comprising a cholesteric matrix permeable to passage of the macromolecular active agent by diffusion.

In consideration of the aforementioned, an implant for delivery of bioactive macromolecule should be simple, so that it will be easy to fabricate and require no follow-up maintenance when implanted. Its size has to be small to avoid imposing excessive tension on the subcutaneous tissue due to stretching when the implant is inserted. The device should hold a sufficient amount of the bioactive macromolecule to sustain the desired effect for many weeks. As well, the excipient component should be absorbable by the body without adverse effect, so that no time consuming surgical procedure is needed to explant the depleted device later. Further, when the implant stopped functioning after a period of time, the incorporated active ingredient should be essentially depleted, so that a new absorbable implant can be inserted, if desired, to continue the regimen without interference from residual activity of the previous implant. Finally, it is important that no surge of the active ingredient should occur in the body causing overdose, if the implant is fractured.

It is an object of the present invention to provide absorbable preparations for the sustained delivery of bioactive macromolecule which continues for many weeks.

It is a further object of the present invention to provide preparations containing bioactive macromolecule powder for sustained reduction of hyperglycemia and for growth promotion which satisfy one or more of the above criteria.

The present invention provides preparations containing bioactive macromolecule powder and an essentially water-insoluble compound in the form of powdered lipoidal fatty substances as the excipient which can be absorbed as well as used by the body without adverse effect. In one embodiment, the admixture of the lipid powder with the bioactive macromolecule powder is compressed into a pellet disc or rod, and a portion thereof is implanted subcutaneously to achieve the desired effect for many weeks. In another embodiment, the pellet disc made of a lipid powder and a bioactive macromolecule powder is broken up into chips, and a suitable amount is inserted subcutaneously through the lumen of a large gauge hypodermic needle. In still another embodiment, the steady delivery of bioactive macromolecule is demonstrated in vitro.

Lipid pellets have been used to deliver low molecular weight compounds, such as steroid hormones (Kincl, et al., J. Pharm. Sci., <u style="single">66</u>, (1977), 490) by diffusion which is inversely proportional to molecular weight. Diffusion for macromolecule therein is considered to be insignificant, however, the present invention has found it to be otherwise. These lipoidal substances, being components of all animal cells and tissues, are varieties of water-insoluble compounds. For use in the present invention, lipoidal substances which are solid at room temperature are selected and made into powder form without any other special treatments aside from grinding in a mortar and pestle for a few minutes. Typical of such solid lipids are saturated fatty acids with 12 or more carbon atoms in the linear chain. The preferred fatty acids are lauric, myristic, palmitic, and stearic acids or a combination of these acids or derivatives. Their glycerides, as well as similar esters of their unsaturated equivalents, are also solids and readily available in abundance. However, simple esters of fatty acids and their corresponding alcohols are sometimes waxy substances which may be hardened and ground at low temperature into the powder form. Polar lipids that can be considered as well include phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylinositol, cardiolipin, galactocerebroside or glucocerebroside. Among the solid steroid or steroid-like members of the solid lipids, the carotenes, vitamins D and K, cholic acids, coprostanol, and cholesterol are readily available in the powder form. The preferred steroidal lipid is cholesterol which is practically insoluble in water, can be readily compressed into a coherent mass without any binder, and is not known to have any pharmacological effect in the body or be deleterious to bioactive macromolecule. It is thus also suitable as an excipient to be used in the instant invention.

In a preferred embodiment of the present invention, 8 parts of natural lipoidal powder is admixed thoroughly with 2 parts of a bioactive macromolecule. The admixture is compressed at 5 metric tons into a small pellet disc.

In another embodiment of the present invention, the pellet disc containing a bioactive macromolecule is cut into chips of approximately 1 mm³ in size. These chips are small enough to be pushed into the subcutaneous space through the lumen of a trocar needle, thus avoiding the need for implantation by a skin incision as required in the case of the disc.

In yet another embodiment of the present invention, the sustained delivery of a bioactive macromolecule in compressed admixture with lipid excipient is shown to be readily demonstrated in vitro.

For the water soluble bioactive macromolecule, the pellet discs or the chips thereof may be tested in vitro by immersion in a stirred buffer solution, and the active component delivered therefrom may be analysed to determine the rate. For less soluble bioactive macromolecule such as insulin, the appropriate amount required daily from the pellet disc of the present invention may be determined to some extent by trials in vivo using different sizes of the insulin containing disc. Therefore, if a lesser amount is desired daily, it can be achieved by implanting a small piece cut from the pellet disc. Otherwise, a slightly larger piece may be used to provide a higher daily dose, if required.

The invention is further described by the following specific examples, which are presented as illustrations only.

EXAMPLE 1

An amount of 160 mg of powdered palmitic acid and 40 mg of powdered bovine insulin (24 IU/mg) are first mixed in a 4 cm by 4 cm plastic weighing boat. The mixed powder is then transferred into a 1.5-mℓ capacity plastic vial with cap and a stainless steel bearing ball is added to aid mixing. The capped vial is pressed onto the platform of a vortex mixer (Vortex-Genie Mixer Catalogue number 12-812, Fisher Scientific Co., Toronto) turned to its maximum speed. After 2 min on the mixer, all the well-mixed powder is carefully tapped into the well of the Spex 13-mm die (Spex Industries, Inc., Edison NJ) without the bearing ball. After the plunger component is lowered into the well, the die set is placed in the centre of the hydraulic press (Spex-Carver Model C, Spex Industries, Inc.). A moderate compression of 0.5 metric ton is first applied before the vacuum pump hose is connected to the die set to evacuate its internal chamber. After 2 min of evacuation to remove entrapped air which would make the interior porous and reduce cohesion, the compression is raised to 5 metric tons and held at this level for 5 min. When the vacuum and compression are released, the mid section of the die housing is unscrewed from its base, and the finished pellet disc (13 mm wide and 1.5 mm thick) is pushed from the central well by gently tap ping the exposed plunger stem. The smooth opaque pellet disc is scored in the middle before it is cut into 2 equal pieces over the groove. The 1/2 piece may be similarly sub-divided further to yield smaller segments.

TEST

Male Wistar rats weighing 320 to 380 g each are divided into 3 groups with 1 as healthy control, a second one as diabetic control and a third group of 2 for testing. Each animal in the second and third group is injected with 50 mg streptozotocin/kg body weight by the tail vein to induce diabetes by irreversibly destroying the insulin producing cells in their pancreas (O.P. Ganda, et al., Diabetes, <u style="single">25</u>, (1976), 595). A drop of blood is taken by tail vein puncture from each of the lightly ether-anesthetized animals and smeared evenly over the tip of a Dextrostix® (blood glucose test strip made by Miles Lab. Ltd., Etobicoke, Ontario). Meanwhile, the timer on the Glucometer® (Colorimeter for Dextrostix®, Miles Lab.) is activated, and after 60 sec when the alarm has sounded, the blood layer is thoroughly washed off from the Dextrostix^R. The intensity of the blue color on the tip is a measure of the blood glucose level in the sample and can be determined quantitatively by inserting the developed Dextrostix® into the Glucometer®, which will show the glucose content on its indicator display in mM glucose/ℓ blood. The results show that the healthy control has a range of about 6-11 mM glucose/ℓ, while the diabetics have a value exceeding 22 mM/ℓ, on the day after streptozotocin injection, which is the maximum limit that can be read on the Glucometer®. On the 8th day after the induction of hyperglycemia, 1 of the diabetic animals in the third group is implanted subcutaneously near the abdomen with 1/8 of the standard size pellet disc prepared as just described. A diabetic rat of body weight between 300-400 g requires about 3 IU insulin daily to lower the blood glucose level to the normal range of 6-11 mM/ℓ. In the 1/8 portion of the standard size pellet disc, there is 5.0 mg insulin or a total of 120 IU, which should be sufficient to supply the need for 40 days. The remaining diabetic animal in the third group is implanted subcutaneously with 1/4 of the standard size pellet disc. The 1/4 disc contains enough insulin to reduce hyperglycemia for about 80 days. The blood sugar level for all 3 groups is monitored at convenient intervals henceforth. While the blood sample is being taken from the lightly anesthetized animal, the presence of the implanted discs is checked by palpation and the weight of the animal is also recorded. Food and water are made available to all the animals ad libitum. The blood glucose results are summarized in Table 1 shown below. <tables id="tabl0001" num="0001"><img file="EP0246540B1_D0001.tif" /></tables> As shown in the last 2 columns of Table 1, reduction in the blood glucose level was observed the next day indicating the fast onset of action. The test animals also continued to gain weight when checked at the time of taking blood samples. In the 30-day period, the diabetic animal with implant in the 3rd Group-A gained 57 g, and the other diabetic animal in the 3rd Group-B gained 82 g during the time when the implant was functional for about 40 days. The healthy control animal in the 1st Group gained body weight steadily as expected, but the diabetic control in the 2nd Group lost 45 g after 38 days and it appeared emaciated as well as stunted.

Hyperglycemia recurred on the 32nd day of the calculated 40 day service life of the 1/8 disc implanted in the diabetic rat of the 3rd Group-A. For the 1/4 disc in the 3rd Group-B of another diabetic animal, the preparation implanted functioned well until the 40th day of the calculated 80-day supply of insulin. Since the blood glucose values monitored over the period were lower than the healthy control group, the implants in the 3rd Group of diabetic animals might have received more than the postulated 3 IU/day which would account for the shorter service life actually observed. Since the implant in the animal of the 3rd Group-B was double in size as compared to the other without showing ill effects, the results also demonstrated the margin of safety in the use of the implant. At biopsy to retrieve the implant, no fibrous tissue encapsulation were evident, and the previously distinct edges of the implant made of palmitic acid was found to have been rounded by erosion. In 1 animal, the spent implant had broken into several small pieces. Analyses by solid phase radioimmunoassay showed that there was only trace amount of insulin remaining in the recovered lipid remnant.

EXAMPLE 2

An amount of 150 mg palmitic acid, 20 mg glyceryl tripalmitate, 25 mg porcine insulin (24 IU/mg), and 5 mg bovine insulin are mixed thoroughly in the plastic weighing boat, and then in a capped polyethylene tube on the vortex mixer. The fine powder admixture is compressed into a standard size pellet disc essentially as described in Example 1, except at 3 metric tons for 5 min. The smooth pellet disc is cut on a Teflon® slab into chips of about 1 x 1 x 1 mm³. The chips are picked up individually with a pair of tweezers and dipped in water to wash off any adhering powder. Several pieces of the chip are transferred to a plastic weighing boat and about 25 mg is taken for subcutaneous insertion near the abdomen of a diabetic Wistar rat (body weight: 350 g) through a trocar needle. The weight of the chips corresponds to about 1/8 of the original standard sized pellet disc and should have an insulin content of about 4 mg. This amount of insulin has a total activity of about 100 IU. At a demand of about 3 IU/day, it is expected to reduce hyperglycemia in the diabetic rat for 33 days. The test animal is bled according to the schedule shown in Table 2, and the Glucometer® method is used to determine the glucose level as in Example 1. <tables id="tabl0002" num="0002"><img file="EP0246540B1_D0002.tif" /></tables> The results obtained show that even when the pellet disc is sub-divided into fragments, there is no unexpected overdose of the incorporated insulin. The same hyperglycemia reduction is again achieved in comparison to the larger pieces of the pellet disc as described in Example 1. However, the duration of maintenance is still consistent with the data for the 1/8 pellet disc given in Table 1 of Example 1. Further, the small difference does indicate the complexity of the on-going process involving erosion, as well as insulin availability, demand, absorption and its inactivation which apparently vary among the test animals. In addition, the more expensive porcine insulin used herein as the major active component is known to be less immunogenic than the similar bioactive macromolecule of bovine origin, and perhaps less prone to complexing with antibodies. At biopsy on day 40, only small round remnants of the inserted chips could be found, indicating absorption had occurred. Further, this animal grew 117 g during the 30-day period of therapy.

EXAMPLE 3

Essentially as already described, a standard size pellet disc is made by compression of a thorough admixture of 130 mg palmitic acid, 30 mg cholesterol, and 40 mg of the porcine growth hormone somatotropin. The pellet disc is cut into 1/2 and both pieces are dropped into a flask containing 1 ℓ bicarbonate buffered solution (pH 8) having 0.1% sodium azide added as the preservative. The solution is vigorously stirred, and 0.5-mℓ aliquots are withdrawn twice weekly for analyses of protein content by colorimetry. The increasing amount of somatotropin in the solution is shown in Table 3 below. <tables id="tabl0003" num="0003"><table frame="all"><title>Table 3</title><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="center">Cumulative Amount of Somatotropin in Solution Derived from Pieces of Pellet Disc</entry></row><row><entry namest="col1" nameend="col1" align="center">Duration (days)</entry><entry namest="col2" nameend="col2" align="center">Hormone in Solution (mg/l)</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">1</entry><entry namest="col2" nameend="col2" align="char" char=".">1.1</entry></row><row><entry namest="col1" nameend="col1" align="right">3</entry><entry namest="col2" nameend="col2" align="char" char=".">3.8</entry></row><row><entry namest="col1" nameend="col1" align="right">5</entry><entry namest="col2" nameend="col2" align="char" char=".">6.0</entry></row><row><entry namest="col1" nameend="col1" align="right">8</entry><entry namest="col2" nameend="col2" align="char" char=".">9.2</entry></row><row><entry namest="col1" nameend="col1" align="right">10</entry><entry namest="col2" nameend="col2" align="char" char=".">10.9</entry></row><row><entry namest="col1" nameend="col1" align="right">12</entry><entry namest="col2" nameend="col2" align="char" char=".">13.0</entry></row><row><entry namest="col1" nameend="col1" align="right">15</entry><entry namest="col2" nameend="col2" align="char" char=".">17.5</entry></row><row><entry namest="col1" nameend="col1" align="right">17</entry><entry namest="col2" nameend="col2" align="char" char=".">18.2</entry></row><row><entry namest="col1" nameend="col1" align="right">19</entry><entry namest="col2" nameend="col2" align="char" char=".">21.1</entry></row><row><entry namest="col1" nameend="col1" align="right">22</entry><entry namest="col2" nameend="col2" align="char" char=".">25.0</entry></row><row><entry namest="col1" nameend="col1" align="right">24</entry><entry namest="col2" nameend="col2" align="char" char=".">26.1</entry></row><row><entry namest="col1" nameend="col1" align="right">26</entry><entry namest="col2" nameend="col2" align="char" char=".">27.4</entry></row><row><entry namest="col1" nameend="col1" align="right">29</entry><entry namest="col2" nameend="col2" align="char" char=".">31.3</entry></row><row><entry namest="col1" nameend="col1" align="right">31</entry><entry namest="col2" nameend="col2" align="char" char=".">34.9</entry></row><row><entry namest="col1" nameend="col1" align="right">34</entry><entry namest="col2" nameend="col2" align="char" char=".">36.5</entry></row><row><entry namest="col1" nameend="col1" align="right">36</entry><entry namest="col2" nameend="col2" align="char" char=".">36.1</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">40</entry><entry namest="col2" nameend="col2" align="char" char=".">36.4</entry></row></tbody></tgroup></table></tables>

The results showed that &gt;90% of the polypeptide hormone entered the stirred solution gradually for a period of over 4 weeks. If more somatotropin is required daily, its content in the pellet disc can be slightly increased. For implantation to promote growth, no antigenic problem will develop if the preparation is used in an isogenic recipient, especially when the excipient components chosen are natural lipid materials present in all tissues.

From the preceding examples, it is thus seen that the objects set forth above are efficiently attained. Since changes may be made in carrying out the above described process and in the article set forth without departing from the scope of the invention, it will be understood that the above examples are illustrative only.