Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/28—Insulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M31/00—Devices for introducing or retaining media, e.g. remedies, in cavities of the body
  • A61M31/002—Devices for releasing a drug at a continuous and controlled rate for a prolonged period of time
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis

Definitions

• the present invention relates to a chemical method for the con ⁇ trolled release and delivery of biologically active substances to body fluids.
• transplanted tissue provides a ready source of insulin whose release is controlled primarily by the level of glucose in the blood.
• This approach has only succeeded with highly inbred strains of animals, however, since tissue rejection otherwise be- comes a limiting factor. Obtaining sufficient quantities of accept ⁇ ably pure islet tissue for future clinical applications also poses significant problems.
• Chick (Science, 187, 847 (1975)) avoids the problem of tissue rejection by culturing pancreatic islets on the outside of semiper- eable tube-shaped membranes, bundles of which are enclosed by a cy ⁇ lindrical shell. Blood flows through the inner tubes when the sys ⁇ tem is interposed into the circulation of an animal providing nutri ⁇ ents to the cells. Glucose and insulin pass across the membrane, while larger immunologically active substances remain inside. The use of cultured tissue however implies that the system is only viable for short periods of time. Mechanical problems, such as clogging of the inner tubes by blood also prevent its extensive use.
• Soeldner et al (NIH publication No. 76-854 (1976), pp. 267)de-scribe a glucose, sensitive electrode which utilizes immobilized glu ⁇ cose oxidase to produce electric current inversely proportional to the blood glucose level.
• Glucose oxidase is a very unstable enzyme in this system, and thus limits the use of such an electrode.
• Another object of the invention is to provide a safe and reliable feedback-controlled release drug delivery system. Another object of the invention is to provide a drug release system which is internally controlled. Still another object of the invention is to provide a controlled drug release system for hormone deficiency diseases.
• a further object of the invention is to provide a method for periodically and control1ably delivering drugs, especially hormones, to human biological fluids.
• a process for feedback controlled delivery of a biological ⁇ ly active substance to an animal body fluid which comprises contact ⁇ ing said fluid with a reversible complex of a conjugate (1) and a binding macromolecule (2), wherein said conjugate (1) comprises a biologically active portion which is intended to be proportionately released into a body fluid stream in response to varying concentra ⁇ tion levels of a component of said body fluid stream, and a complex- ing substrate portion which conjugates with said biologically active portion and which is characterized by affinity to said binding macro ⁇ molecule (2) competitively or non-competitively with said variable component of said body fluid; thereby causing the component " present in said body fluid to complex to said binding macromolecule and to release said conjugate (1) therefrom into said fluid.
• the objects of the invention have also been attained by provid ⁇ ing a process for the controlled delivery of a biologically active hypoglycemic substance to a glucose containing human body fluid which comprises contacting said fluid with a reversible complex of (1) a biologically active hypoglycemic substance which is a conjugate be- tween a carbohydrate and a hypoglycemic agent, and (2) a binding macromolecule having affinity for glucose and for the carbohydrate portion of said conjugate (1); thereby causing the glucose present in said fluid to complex to said binding macromolecule and to release said conjugate into said fluid.
• a controlled release medication system which comprises a re ⁇ versible complex of (1) a biologically active hypoglycemic substance which is a conjugate between a carbohydrate and a hypoglycemic agent and (2) a binding macromolecule having affinity for glucose and for the carbohydrate portion of said conjugate.
• Figure 1 is the elution profile from a column of immobilized concanavalin A, of insulin (A) and maltose-insulin conjugate (B) in the presence of glucose as eluant (arrows).
• Figure 2 is the elution profile from a column of immobilized concanavalin A of a maltose-insulin in the presence of pulses of in ⁇ creasing glucose concentration.
• the drug delivery system of the present invention comprises a reversible non-covalent complex (B:A-C) between a binding macromole ⁇ cule (B) and a conjugate of a biologically active substance (A) with a complexing substance (C).
• the conjugate (A-C) contains a biologic ⁇ ally active portion (A) and a complexing portion (C). Equations (1) and (2) show a first embodiment for the operation of the system. This embodiment will be called directly competitive in analogy to the well-known kinetic system of competitive inhibition for enzymes. (Mahler and Cordes, Biological Chemistry, 2d Edition (1971) pp. 295- 297.)
• the complex B:A-C is initially in reversible chemical equilibrium with the separated components B and A-C (eq. 1).
• the binding site of B can only recognize molecules of shape C, either when these are free in solution or when they are coupled to another component such as in conjugate C-A.
• the degree of association of B with conjugate A-C is ideally very high, so that when no or little C is present in ⁇ dependently, the system lies mostly on the B:C-A side of the equili ⁇ brium (1), and there are very few free, unassociated B molecules present in solution (eq. 1).
• the concentration levels of C increase in the system, the molecules of C start competing for the available binding sites on B and the equilibrium represented by equation (2) becomes more important.
• Conjugate A-C in equation (3) is recognized by B although complexing portion C is, in overall structure, different than C.
• the recogni ⁇ tion sites of both C and C molecules are, however, exactly alike.
• C and C bind at the same site and displace each other from the site.
• a second embodiment of the system which is useful in the present invention is one wherein the displacement of conjugate A-C (or A-C), or more precisely, the blocking of the available site on B for its complexation therewith occurs non-competitively.
• B has two sites, one of which complexes which conjugate C-A or C-A, and the other one with free C.
• the binding sites are structurally different but they interact by " negative feedback.
• Binding macromolecule B can by any of a number of well-known macromolecular entities which exhibit molecular recognition and re ⁇ versible binding of other micro- or macromolecules.
• B is preferably a natural binding protein, such as an antibody, an enzyme, a regula ⁇ tory protein, a drug receptor site or the like.
• synthetically modified binding molecules such as chemically modified proteins. Such modified proteins sometimes have increased or decreased affinities for their substrates when compared to their natural unmodified counterparts.
• macromoe cule is also meant a conjugate of a smaller molecule which shows binding characteris- tics, with a larger polymeric molecule which, by itself does not.
• binding proteins B antibodies and lectins are the most preferred.
• Antibodies can be prepared and purified from animals in standard fashion (Eisen, H.N. "Immunology", Harper & Row, 1974). Antibodies have the advantage of being induceable in an animal by challenge with an appropriate anti genie agent. Since this agent can be chosen from any chemical family, e.g., amino acids, carbohydrates, their respect ⁇ ive polymeric derivatives, or the like, the resulting antibodies may have a wide range of binding specificity and affinity.
• Lectins are also particularly useful in this invention.
• Lectins are carbohy ⁇ drate-binding proteins of plants and animals with a wide variety of specificities for carbohydrates, (Lis et al, Ann. Review of Biochem ⁇ istry, 42, 541 (1973); I.J. Goldstein and C. E. Hayes, Adv. in Carbo ⁇ hydrate Chemistry and Biochemistry, Vol. 35, R.S. Tipson and D. Hor- ton, eds. (Academic Press, New York, 1978, pp. 128-341), herein in ⁇ corporated by reference).
• concanavalin A a Jack Bean lectin
• soybean lectins are specific for ⁇ and ⁇ -D-N-acetylgalactosamine and -D-galactose units
• wheat germ lectin is specific for ⁇ -D-N-ace- tyl glucosamine.
• a manno-oligosaccharide containing from 2 to 7 (1, 2) -D-mannopyranosyl residues is used as the carbohydrate.
• the molecular complexation reaction between binding macromole ⁇ cule B and molecule C, or the C or C portion of conjugate A-C, has to be reversible and non-covalent.
• This bonding between B and C (or B and A-C) is caused by non-covalent forces such as hydrophobic, ion- ic, hydrogen bonding forces and the like.
• non-covalent forces such as hydrophobic, ion- ic, hydrogen bonding forces and the like.
• the functional relation between component C present in the body fluid and the biologically active agent A is very important.
• the biologically active conjugate A-C (or A-C) is released in propor ⁇ tionate amounts to the levels of component C present in the body fluid.
• Agent A therefore can be a hormone-type substance which directly regulates the levels of C in the fluid, by for example, caus ⁇ ing a decrease of the levels of C.
• Agent A need not be directly functionally related to component C but only indirectly. In such case, increasing levels of C in the fluid would release con ⁇ jugate A-C (or A-C) which then, via its biologically active portion A, will act on a biological system unrelated or only indirectly re ⁇ lated to component C.
• A-C is a hormone or hormone-like substance and C is a metabolite participating in a- feed ⁇ back regulatory loop with A.
• Hormones are substances secreted by endocrine glands or in tis ⁇ sues and released into the bloodstream, " by which they are transported to other tissues where, by selectively binding to specific receptors, they exert their effects. Hormones possess a wide range of chemical structures: peptides, steroids, derivatives of amino acids and der ⁇ ivatives of fatty acids. Hormones are glandular or tissue-originat ⁇ ed.
• the common glandular hormones useful with the system of the present invention are the pituitary and hypothala ic polypeptide hormones such as oxytocin, arginine-vasopressin, lysin-vasopressin, adrenocorticotropin (ACTH), follicle-stimulating hormone (FSH); soma- totropin (GH), luteinizing hormone (LH), melanocyte-stimulating hor ⁇ mone (MSH), prolactin, thyroid-stimulating hormone (TSH); the para- thyroid hormone; calcitonin; the hormones of the pancreas such as insulin; glucagon and somatostatin, the adrenal cortical hormones such as mineralocorticoids as aldosterone and desoxycorticosterone, glucocorticoids as cortisone and hydrocortisone, sexual hormones as androgens, anabolic steroids, estrogens, progestagens.
• the hormones of the pancreas such as
• the coupling molecule C is the substance whose varying levels will proportionately release active conjugate A-C (or A-C).
• C is a fluid component whose levels are reg- ualted by A-C. In such case, C has to be in functionnal relation to
• C is chosen so that a feedback loop exists be ⁇ tween C and A; i.e., when the levels of C increase in the blood, the levels of A increase under normal circumstances, thus returning the level of C, and in turn A, to their equilibrium positions.
• Examples of coupling molecules C are: glucose (regulated by insulin and also by other hypoglycemic agents such as sulfonylureas and bi- guanides); calcium (regulated by calcitonin); cortisone(regulated by ACTH); progesterone (regulated by LH); testosterone (regulated by LH); thyroid hormones (regulated by TSH); prolactin (regulated by pro!actin-release inhibiting hormone); growth hormone (regulated by growth hormone releasing-inhibiting hormone); electrolytes (regulat- ed by mineralocorticoids).
• glucose regulated by insulin and also by other hypoglycemic agents such as sulfonylureas and bi- guanides
• calcium regulated by calcitonin
• cortisone regulated by ACTH
• progesterone regulated by LH
• testosterone regulated by LH
• thyroid hormones regulated by TSH
• prolactin regulated by pro!actin-release inhibiting hormone
• growth hormone regulated by growth hormone
• the coupling between A and C can be carried out directly by us ⁇ ing appropriate functional groups on both molecules or by using a bridging or spacer group between them.
• Nucleophilic groups on one component such as free -OH or -NH 2 or -SH can be reacted with elec- trophilic groups on the other component, such as esters, ketones, al ⁇ dehydes and the like, to give covalent adducts.
• a reducing agent such as NaBH ⁇ , KBH ⁇ , Na CNBH 3
• Another possible coupling react- ion is that between free amines on proteins and ester functions on proteins or carbohydrates, to give amides.
• the naturally occurring free carboxylic acids of the protein or carbohydrate have to be chemically esterified first, as by react ⁇ ion with a diazoalkane such as diazomethane.
• the final bond between A and C has to be stable and irreversible and is, most preferably, covalent.
• the properties of the conjugate A-C or A-C are such that it should retain both the reversible affinity and binding characterist ⁇ ics of C and the biological activity of A. Its properties are there ⁇ fore a composite of those of both its constituents.
• the choice of appropriate coupling partners and coupling methods are critical. Chemical attachment of the type required by the present invention might lead to a great or even total loss of binding or of regulatory activity, or both. It is crucial that none of these activities be lost after coupling.
• the B:A-C (or B:A-C) complex is brought into intimate contact with the physio ⁇ logical human fluid to be treated.
• the controlled release of active conjugate A-C (or A-C) begins when the amount of free C in the fluid contacting the complexes reach a given critical high level. Virtu ⁇ ally no active conjugate is released when the levels of free C fall below the critical level. It is also possible to simultaneously con ⁇ tact the body fluid with complexes between B and active conjugates A-C, A-C" and A-C" wherein these conjugates have different affinity towards B. In this way, increasing levels of C in the fluid would release a steadily increasing amount of active conjugate.
• Still another possibility is to contact the body fluid with complexes be ⁇ tween B and A-C, A'-C and A"-C, where A, A' and A" represent biolo ⁇ gically active portions of the conjugate exhibiting different biolo ⁇ gical activities.
• A, A' and A" represent biolo ⁇ gically active portions of the conjugate exhibiting different biolo ⁇ gical activities.
• increasing levels of fluid compon ⁇ ent C will release two or more different substances which exhibit different biological effects.
• the system of the present invention is ideally suited for use in externally or internally implanted devices, such as insulin pumps or the like.
• the complex B:A-C may be present in a serniperme- able or porous tube or fiber, which is brought into contact with hu ⁇ man fluid such as plasma.
• the pores of the tube or device are chos ⁇ en so that the complex and the binding molecule B cannot pass there ⁇ through and are "trapped" inside.
• the pores however are permeable to conjugate A-C and free component C.
• conjugate A-C (or A-C) is released into the plasma.
• Another embodiment of the in ⁇ vention is to fix the binding macromolecule B covalently either di ⁇ rectly, or via a- spacer or bridging group, to the tube itself.
• the techniques of solid-state immobilization of enzymes and other pro- teins on resins, films, test tubes, glass beads and the like are well known (see e.g., Zaborsky, C.
• this invention relates to a system for the delivery of active hypoglycemic agents, prefer ⁇ ably insulin, to the bloodstream of patients with diabetes mellitus.
• Diabetes mellitus is a hereditary disease characterized by the rela ⁇ tive or absolute deficiency of insulin.
• Insulin is a polypeptide hormone made of 2 chains of amino acids, molecular weight 6000. It is synthesized, stored and released from the pancreas primarily in response to blood glucose levels. Insulin carries out a variety of physiological functions such as for example, activation of transport systems for glucose and of certain enzymes involved- in increased glu- cose utilization, glycolysis, glycogenesis and lipogenesis. Absence of insulin thus causes an increase in the blood levels of glucose (hyperglyce ia) . • u ⁇ - i -A
• A is an active hypo ⁇ glycemic agent
• C is glucose or an actively binding analogue thereof with complexing characteristics similar to those of glucose
• B is a carbohydrate-binding molecule
• the oligosaccharide- insulin conjugate binds to a lectin such as concanavalin A and can be displaced therefrom by solutions with increasing glucose content.
• a lectin such as concanavalin A
• Various hypoglycemic agent-conjugates complementary to the major combining sites of the lectins, can be synthesized and the products are stable. Since lectin binding affinity for various saccharides varies more than ten-fold, a mixture of different high- and low-af ⁇ finity semisynthetic conjugates can be combined to produce clinically desirable elution profiles.
• a number of active insulin derivatives have been prepared (Ellis, M.S. et al, Diabetologia T : 340, 1975; Tompkins, C.V.
• Another application is the preparation of glycosylated derivatives of somatostatin. This would provide glucose-controlled inhibition of the excessive gluca- gon secretion which is thought to exacerbate diabetic hyperglycemia.
• Yet another application of the system in a mode where agent A is not a hormone involved in the regulation of component C, is for example the release of enzymatic inhibitors in diabetes mellitus. Studies in animals (Winegrad, A.I, et al , In Proceedings of the VII Congress of the International Diabetes Federation, ed.. W.J. Malaisse and J. Pirart, pp.
• GBM glomerular basement membrane
• Still another application of the present inveniton is in the * simultaneous release of a combination of hypoglycemic agents.
• Alburn et al USP 3,912,807 have shown that the combination of somatostatin and insulin is very effective in the treatment of diabetes mellitus.
• Dietze et al USP 4,150,121 have demonstrated that kinin-insulin combinations are useful in diabetes, and Laborit (USP 4,035,486) has shown that guanosine increases glucose utilization in diabetic ani ⁇ mals treated with insulin or acetylcholine.
• the system of the pre- sent invention can thus be simply adapted to the glucose-dependent release of drug combinations.
• a first one comprising a lectin and a conjugate of carbohydrate and insulin
• a second one comprising a lectin and a conjugate of carbohydrate and guanosine.
• raising glucose levels will release both guanosine and insu ⁇ lin into the body fluid.
• the insulin conjugate was stored at 4°C and was stable for at least seven weeks regardless of whether or not reducing agent had been added.
• II. Bioactivtty of Maltose-Insulin Conjugate The conjugate prepared in I was tested using the blood-glucose depression assay modified from the U.S. Pharmoacopeia (Vol. XVIII, 883-884 (1967)). Preparations varied from 78 to 95% of unmodified insulin bioactivity. The results are given in Table 1.
• Figure 1 shows the elution profile obtained from 20 mg of un ⁇ modified insulin (A) and maltose-insulin (B) applied to columns (24.0 X 1.5 cm I.D.) of immobilized Concanavalin A.
• Initial buffer was 1 mM MgCl 2 , 1 mM CaCl 2 , 50 mM NaCl , 25 mM sodium phosphate, pH 7.4.
• 0.2 M glucose in the col ⁇ umn buffer was begun.
• Fractions (2.0 ml) were collected at a flow rate of 10-15 ml/h. Unmodified insulin did not bind to Concanavalin A.
• FIG. 1 shows the elution profile obtained from 20 mg maltose- insulin derivative applied to a column (12.0 X 1.5 cm I.D.) of immo ⁇ bilized Concanavalin A.
• Initial buffer was 1 mM MgCl 2 , 1 mM CaCl 2 , 50 mM NaCl, 25 mM sodium phosphate, pH 7.4.
• Displacement of hormone was accomplished using 1.0 ml pulses of column buffer containing Varying amounts of glucose. Glucose content is indicated over the appropriate arrows. Fractions (1.7 ml) were collected at a flow rate of 10-15 ml/h. Glucose-pulse experiments with the lectin- bound maltose-insulin derivative demonstrate that hormone release is a function of the quantity of glucose present.

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• 1979
  • 1979-07-31 US US06/062,484 patent/US4348387A/en not_active Expired - Lifetime
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