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WO1997009061A1 - Treatment of diabetes via administration of hormonally ineffective insulin - Google Patents

Treatment of diabetes via administration of hormonally ineffective insulin Download PDF

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Publication number
WO1997009061A1
WO1997009061A1 PCT/US1996/014348 US9614348W WO9709061A1 WO 1997009061 A1 WO1997009061 A1 WO 1997009061A1 US 9614348 W US9614348 W US 9614348W WO 9709061 A1 WO9709061 A1 WO 9709061A1
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insulin
hormonally
diabetes
ineffective
patient
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PCT/US1996/014348
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French (fr)
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Yi Wang
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Alexion Pharmaceuticals, Inc.
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Publication of WO1997009061A1 publication Critical patent/WO1997009061A1/en

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    • 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

Definitions

  • Diabetes Mellitus Diabetes mellitus is the most common endocrine disease, and is characterized by abnormalities of glucose metabolism.
  • the abnormal glucose metabolism associated with this disease results in hyperglycemia (high blood glucose levels) and eventually causes complications of multiple organ systems, including eyes, kidneys, nerves, and blood vessels.
  • Patients with persistent hyperglycemia or abnormal glucose tolerance are generally diagnosed with the disease, although most commonly patients initially present with excessive urination (polyuria) and frequent drinking due to extreme thirst (polydipsia) . These typical initial symptoms result from the osmotic effects of hyperglycemia.
  • diabetes mellitus is typically associated with pancreatic dysfunction, particularly of the beta cells of the pancreatic islets of Langerhans. This dysfunction may lead to destruction of the islet beta cells, which produce insulin, a glucose regulatory peptide hormone.
  • Diabetes mellitus has been generally categorized as insulin dependent or type 1, versus non-insulin dependent, or type 2. However, this terminology has evolved as the disease has become better understood. For example, it has been found that in some patients suffering from non-insulin dependent diabetes, the disease progresses into an insulin dependent form, while in other patients insulin dependence does not develop.
  • Type 1 autoimmune islet pathogenesis, i.e., to diabetes caused by islet-specific autoimmune attack, and is ⁇ o used hereinafter.
  • IDDM insulin dependent diabetes mellitus
  • pre-IDDM refers to an autoimmune condition that can be detected by biopsy or by analysis of autoimmune responses, in which pancreatic islet beta cells are being subject to a specific autoimmune attack to an extent where some cells may be subject to destruction.
  • pre-IDDM the destruction (if any) has not progressed to an extent sufficient to require the administration of insulin. Since there can be a point in the early stages of type 1 diabetes in which overt symptoms are observed but some islet function remains, not all type 1 diabetes is classified as IDDM, and not all pre-IDDM presents without overt symptoms.
  • the metabolic complications associated with the abnormal metabolism caused by insulin insufficiency can affect numerous organ systems.
  • the most common acute metabolic complication is that of diabetic ketoacidosis, characterized by severe hyperglycemia (and resulting hypovolemia caused by osmotic diuresis) as well as metabolic acidosis induced by excess free fatty acid release and the production of ketone bodies.
  • a patient experiencing these complications may present with anorexia, nausea, vomiting and/or altered consciousness, and must be treated aggressively with insulin therapy as well as fluid and electrolyte replacement.
  • Diabetic retinopathy is a leading cause of blindness, and is initiated by increased permeability of retinal capillaries which can progress to occlusion, hemorrhage, aneurysm formation, and neovascularization known as proliferative retinopathy.
  • kidney and neurological diseases are common complications of diabetes.
  • Diabetic nephropathy causes about half of end-stage renal disease in the United States. Histologically, the nephropathy is characterized by glomerular basement membrane widening and mesangial thickening. Initial signs include increasing proteinuria, with azotemia ultimately leading to renal failure.
  • Diabetic neuropathy can affect any part of the nervous system, with the possible exception of the brain. The neuropathy is most commonly seen a ⁇ peripheral polyneuropathy, with symptoms including numbness, paresthesias, severe hyperesthesias, and pain. Autonomic neuropathy can cause gastrointestinal dysfunction, orthostatic hypotension, bladder dysfunction or paralysis, and impotence.
  • Diabetic foot ulcers represent a special problem of diabetics, and appear to be due primarily to abnormal pressure distribution secondary to diabetic neuropathy.
  • the ulcerous lesions are often worsened by concomitant peripheral vascular disease and infection.
  • meticulous control of blood glucose has been associated with amelioration of the late complications of diabete ⁇ , ⁇ uggesting that therapie ⁇ that preserve beta cell function could reduce or eliminate the majority of the complications of diabetes mellitus.
  • Type 1 diabetes only develops in genetically susceptible individuals, and symptoms generally appear before age 40, with the peak incidence of onset of overt symptomology occurring in the second decade of life.
  • the pathogenesis of type 1 diabetes is characterized by an initial phase of leukocyte ⁇ infiltration into the islets, referred to a ⁇ in ⁇ uliti ⁇ , followed over a period of time by the actual de ⁇ truction of the islet beta cells by autoimmune attack.
  • the insulitis phase is characterized by infiltration of pancreatic islets by both lymphocytes and cells of the monocyte/macrophage lineage, and entail ⁇ both cell- mediated inflammation as well a ⁇ attack by islet-specific cytotoxic antibodies.
  • T Cells The autoimmune destruction of the beta cells of the pancreatic islets in Type 1 diabetes is believed to be initiated by white blood cells (leukocytes), mo ⁇ t importantly T cell ⁇ . T cells, or T-lymphocytes, are mononuclear white blood cells that provide many essential immune functions. The importance of T cells in human autoimmune diseases has been increasingly appreciated in the past decade.
  • T cells mediate tissue injury by indirect and direct means.
  • T cell ⁇ of both CD8 + (cytotoxic) and CD4 + (helper) ⁇ ubset ⁇ secrete a variety of inflammatory cytokines that can damage tissue ⁇ indirectly by activating various other types of white blood cell ⁇ .
  • Example ⁇ of ⁇ uch T cell effects include activation of antibody ⁇ ecreting B cell ⁇ ( ⁇ timulating humoral immune activity) and activation of macrophage ⁇ , which can cau ⁇ e acute ti ⁇ sue damage and inflammation by releasing hydrolytic enzymes, reactive oxygen species, and additional pro-inflammatory cytokines.
  • direct ti ⁇ ue damage can be mediated by CD8 + cytotoxic T cells attacking cells displaying target antigen ⁇ .
  • T cell receptors T cell receptors
  • TCRs membrane bound antibody-like binding structures
  • TCR ⁇ bind with high specificity to particular antigens.
  • antibody-producing cell ⁇ which develop as multitudinous clones of cells, each clone producing antibodies with unique specificities, T cells develop as a vast number of distinct clones, and any particular T cell clone expresses a single type of TCR with a defined binding specificity.
  • T cell clones with TCRs that bind specifically to self antigens are respon ⁇ ible for the development of autoimmune di ⁇ eases.
  • a particular polypeptide antigen typically comprises numerous ⁇ ubmolecular features, known as epitopes, that each can serve a ⁇ a di ⁇ tinct binding site for a particular antibody or TCR.
  • T cell clones reactive with various epitopes of a small number of autoantigens, become activated and are involved in pathogenesis. Even in individuals suffering from autoimmune diseases, only a small subset of T cell clones (0.1-1%) are known to recognize autoantigens.
  • Variou ⁇ mechanism ⁇ have been po ⁇ tulated to play a role in the pathogenic activation of disea ⁇ e-cau ⁇ ing autoreactive T cell ⁇ .
  • Primary activation of antigen presenting cells (APCs) by infection or local inflammation is implicated in one such mechanism.
  • APCs activated in this way can then provide powerful co-stimulation for hitherto unreactive T cells.
  • T cell-mediated autoimmune disease In addition to external factors, underlying the emergence of all T cell-mediated autoimmune disease is a complex pattern of inherited susceptibility determined by multigenic factors. For further di ⁇ cus ⁇ ions of these various factors, Steinman, 1995, reviews current theories of autoimmunity.
  • Apoptosi ⁇ thu ⁇ play ⁇ a large role in shaping and maintaining the T cell repertoire and contributes to the establishment of self- tolerance by actively eliminating cells expressing autoreactive TCRs.
  • T cells are sensitive to apoptotic cell death induced by a variety of stimuli at multiple points in their life ⁇ pan (see, for example, Lenardo 1991; Boehme and Lenardo 1993; Critchfield et al. 1994) .
  • Positive selection factors are also believed to play a role in regulating the survival of specific T cell clones.
  • the reduction or expansion of the number of individual T cells of a particular clone in an organism by these and other mechanisms serve to modulate the responsivenes ⁇ of the organi ⁇ m's immune sy ⁇ tem to a particular antigen.
  • Apoptosis occurs in many biological sy ⁇ tem ⁇ (see, for example, Kerr et al. 1991; Lock ⁇ hin and Zakeri, 1991; Cohen et al. 1992; Duvall and Wyllie, 1986; Cotter et al. 1990) .
  • a cell undergoing apopto ⁇ i ⁇ undergoe ⁇ a ⁇ pecific program of event ⁇ -- cellular and biochemical proce ⁇ e ⁇ that depend upon active metaboli ⁇ m and contribute to the cell's self-de ⁇ truction.
  • apoptotic T cell ⁇ In apoptotic T cell ⁇ , the nucleus shrinks, the chromatin condenses, the genetic material (DNA) progressively degrades into ⁇ mall (nucleo ⁇ omal repeat ⁇ ized) fragment ⁇ , there i ⁇ cytopla ⁇ mic compaction, the cell membrane forms blebs, and the cell eventually collapses (Kawabe and Ochi, 1991; Smith et al. 1989) . Cells cannot recover from apoptosis, it results in irreversible cell death (Kawabe and Ochi, 1991; Smith et al. 1989) .
  • TNF-related cytokine known as the FAS ligand and its receptor, CD95 (the FAS receptor)
  • CD95 the FAS receptor
  • T cells that do not undergo apoptosis, but which have become activated will carry out their "effector" functions by causing cytolysis, or by secreting lymphokine ⁇ that cau ⁇ e B cell response ⁇ or other immune effect ⁇ (Paul, 1989) .
  • effector functions are the cause of tissue damage in autoimmune and other diseases.
  • Type 1 diabetes is con ⁇ idered to be mediated by T cell ⁇ .
  • the di ⁇ ease is believed to be a consequence of inappropriate ⁇ pecific T cell responses to certain islet beta cell proteins that act as autoantigens.
  • autoantibodies against variou ⁇ self antigens have also been reported in IDDM patients.
  • the antigens reported to be bound by these autoantibodie ⁇ include many of those that have been reported to be recognized by autoreactive T cells.
  • Autoantigens that are subject to autoimmune respon ⁇ es in Type 1 patients include ⁇ ialyglycolipid; the 64kDa and 67kDa GAD (glutamate decarboxyla ⁇ e) autoantigen ⁇ ; insulin; a 38kD antigen from the secretory granule ⁇ of beta cells; an antigen cros ⁇ reactive with antibodies to bovine albumin known a ⁇ the beta cell p69 protein, PM-1, or di ⁇ ease-modifying antigen, a beta cell cyto ⁇ keletal protein known as peripherin, gluco ⁇ e tran ⁇ porter proteins, including GLUT-2 ,* heat shock protein 65 (HSP 65) , including the p277 peptide; carboxypeptida ⁇ e H; a 52Kd molecular mimic of Rubella viru ⁇ antigen; a beta cell membrane a ⁇ sociated protein of 150kDa; a protein antigen located at the secretory pole of the rat insulinoma cell line RINm38, referred
  • modulation of insulin reactive T cells would be closing the barn door after the horses had gone, the anti-insulin reactions being observed so late in disease progression that their modulation would not be expected to affect the onset or severity of di ⁇ ea ⁇ e.
  • T cells also respond to other islet cell antigens.
  • the ⁇ e include a 38kD antigen from the ⁇ ecretory granule ⁇ of beta cells, and serum albumin.
  • heat shock protein 65 has been implicated based upon the fact that HSP-specific T cells were shown to transfer disease in NOD mice.
  • Carboxypeptidase H i ⁇ a molecule found in islet secretory granules and is associated with the production of peptide hormone ⁇ and neurotran ⁇ mitter ⁇ . It was identified as a potential islet autoantigen by the screening of cDNA expression libraries with sera from IDDM or pre-IDDM patient ⁇ .
  • ICA12 and ICA512 have also been identified by screening of cDNA expression libraries.
  • Insulin autoantibodies can be detected in around 50% of new onset patients, and are highly associated with islet cell autoantibodies (ICA) and the HLA-DR4 phenotype.
  • ICA islet cell autoantibodies
  • T cell responses to insulin as autoantigen have al ⁇ o been de ⁇ cribed.
  • cellular responses to human insulin were present in almost 90% of ICA-positive first degree relatives of IDDM patients.
  • insulin reactive T cells from diabetic NOD mice can tran ⁇ fer diabetes to non-diabetic NOD mice.
  • Type 1 Disease As discus ⁇ ed above, there i ⁇ a genetic aspect to the incidence of type 1 diabetes. Accordingly, individuals with a known family history of the di ⁇ ea ⁇ e can be monitored for early, preclinical ⁇ igns of disease development, e.g., by monitoring levels of the autoantibodies discu ⁇ sed herein. In addition, genetic tests can identify certain individuals at increased ri ⁇ k of developing the disease (see, for example, Walston et al . 1995) .
  • Prediction of type 1 diabetes may also be facilitated by monitoring of the subject's blood sugar levels, preferably, in conjunction with the administration of a glucose tolerance test to the subject. Such procedures are preferably carried out in combination with the monitoring of titers of the subject's circulating autoantibodies, such autoantibodies selected from the group consi ⁇ ting of IAA, ICA, and GAD ⁇ pecific autoantibodie ⁇ .
  • the first line of treatment is diet, with appropriate caloric intake based on ideal body weight and a defined distribution among protein, glucose, and fat.
  • the most important component of therapy is the administration of insulin, the goal of which is to maintain glucose levels as clo ⁇ e to the normal range a ⁇ possible throughout the day.
  • Insulin is available in rapid, intermediate, and long-acting formulations which vary in onset, peak, and duration of action, and can be u ⁇ ed in varying ⁇ chedule ⁇ of admini ⁇ tration to attempt to optimally regulate plasma glucose levels.
  • Intensive insulin therapy refer ⁇ to a rigorou ⁇ regimen of admini ⁇ tration of hormonally effective in ⁇ ulin and monitoring of blood ⁇ ugar levels.
  • Thi ⁇ regimen is designed to control blood gluco ⁇ e as precisely as pos ⁇ ible.
  • the re ⁇ ult ⁇ of the multicenter Diabetes Control and Complication Trial established that complications of diabete ⁇ are ⁇ ignificantly diminished by better control of blood glucose level ⁇ , and thu ⁇ demon ⁇ trated the desirability of intensive insulin therapy.
  • intensive insulin therapy requires a high level of patient awareness and compliance, a ⁇ well as a highly skilled care team of physicians, nurses, and dietitians. The goals of intensive insulin therapy are thus extremely difficult to achieve, even with motivated and educated patients.
  • Another problem is that a higher rate of hypoglycemia is seen in such rigorously treated patients than in patient ⁇ receiving standard, less rigorous, in ⁇ ulin regimen ⁇ .
  • the Diabetes Control and Complication Trial highlighted not only the benefit to overall metabolic health of maintaining normal blood glucose levels, but al ⁇ o a fundamental problem a ⁇ ociated with the treatment of type 1 diabetes, namely that the overt symptoms of the disease are manifested only when essentially all of the patients' islets are destroyed.
  • Oral agents for diabetes such a ⁇ the sulfonylureas, act primarily by stimulating the release of insulin from dysfunctional beta cells, and thus are not useful for most patients with type 1 disease, i.e. for those patients with IDDM.
  • a major unmet goal in the treatment of diabete ⁇ has been to develop a therapy capable of aborting the autoimmune attack on the islet beta cells prior to their complete destruction, thereby preserving enough endogenous function to maintain normal metabolic control. Induction of tolerance.
  • NOD non-obe ⁇ e diabetic
  • mou ⁇ e model of diabete ⁇ it has been shown that oral feeding of insulin delayed the onset and reduced the severity of the disease.
  • the mechanism proposed to explain oral tolerance is that oral antigen administration induce ⁇ populations of antigen-specific Th2 T cells that secrete antiinflammatory cytokines such as IL-4, IL-10, and TGF-beta.
  • T cells circulate and are activated to secrete cytokines only in the presence of their ⁇ pecific antigen. Thu ⁇ , in ⁇ ulin- specific Th2 T cells would be activated only in the pancreas where they would produce ⁇ uppre ⁇ ive cytokines to modulate the autoimmune process. This mechanism does not require, therefore, that the oral antigen actually represent a disea ⁇ e-specific autoantigen, but rather only that it is expressed in a tis ⁇ ue specific fashion.
  • method ⁇ de ⁇ igned to produce T cell tolerance require the identification of the actual disea ⁇ e-specific autoantigens that are targeted by autoimmune attack. Such antigens are then admini ⁇ tered to patient ⁇ in an appropriate tolerizing fashion (which may al ⁇ o induce non-antigen-specific tolerizing effects) .
  • type 1 diabetes is in significant measure a disea ⁇ e mediated by islet-specific autoreactive T cells, therapy of this type ⁇ hould be fea ⁇ ible in principle. Thu ⁇ , induction of neonatal tolerance to GAD, a ⁇ referred to above, prevented on ⁇ et of di ⁇ ea ⁇ e in NOD mice.
  • Apopto ⁇ i ⁇ is a form of programmed cell death that occurs in many biological systems (Kerr et al. , 1991. Apoptosis: The molecular basi ⁇ of cell death, Tomei and Cope (eds.), Cold Spring Harbor Laboratory Press, Plainview, New York, pp. 5; Lock ⁇ hin and Zakeri, 1991. supra, pp. 47; Cohen et al . , 1992. Ann Rev Immunol 10, pp. 267; Duvall and Wyllie, 1986. Immunol Today 7, pp. 115; Cotter et al. , 1990. Anticancer Research 10, pp. 1153) .
  • apoptotic cell undergoes a ⁇ pecific program of event ⁇ that depend upon active metabolism and contribute to its own self-destruction.
  • the nucleus shrinks, the genetic material (DNA) gradually ⁇ ively degrade ⁇ , and the cell eventually collap ⁇ e ⁇ (Kawabe and Ochi, 1991. Nature 349, pp. 245-248; Smith et al. , 1989. Nature 337, pp. 181-184) .
  • Cell ⁇ cannot recover from apopto ⁇ is, it result ⁇ in irrever ⁇ ible killing (Kawabe and Ochi, 1991. Nature 349, pp. 245-248; Smith et al., 1989. Nature 337, pp. 181-184) .
  • T cell ⁇ that do not undergo apopto ⁇ i ⁇ , but which have become activated will carry out their "effector” function ⁇ by cau ⁇ ing cytoly ⁇ i ⁇ , or by ⁇ ecreting lymphokines that cau ⁇ e B cell responses or other immune effects (Paul, 1989. Fundamental Immunology, 2nd Ed. Paul (ed.), Raven Pres ⁇ , New York, pp. 3-38) .
  • effector functions are the cau ⁇ e of ti ⁇ ue damage in autoimmune and other di ⁇ ease ⁇ .
  • a powerful approach to avoiding disease is thus to permanently eliminate by apoptosis only those T cells reactive with autoimmune disease-inciting antigens, while leaving the majority of the T cell repertoire intact.
  • Figure 1 shows the results of the CYTOXAN induced IDDM experiments .
  • Figure 2 show ⁇ the re ⁇ ult ⁇ of the adoptive tran ⁇ fer of IDDM experiment ⁇ .
  • the objects of this invention are to provide novel immunomodulatory methods for both the prevention and treatment of type 1 diabetes mellitus.
  • the invention provides a method of treating a patient in need of such treatment, e.g., a patient selected from the group of patients consisting of patients at risk of developing diabetes and patients suffering from diabetes (particularly early in disease progression before all of the patient's beta cells have been destroyed by the disease), so as to delay the onset or reduce the symptoms of diabetes in the patient.
  • This method comprises the administration of do ⁇ e ⁇ containing mea ⁇ ured amounts of an adjuvant free hormonally ineffective insulin related polypeptide to the patient.
  • thi ⁇ admini ⁇ tration is carried out on a therapeutic T cell modulatory schedule, i.e., a schedule designed to induce apoptosis, anergy, or other modulation of the autoimmune activity of T cells reactive with at least one epitope of the hormonally ineffective insulin related polypeptide.
  • a therapeutic T cell modulatory schedule i.e., a schedule designed to induce apoptosis, anergy, or other modulation of the autoimmune activity of T cells reactive with at least one epitope of the hormonally ineffective insulin related polypeptide.
  • the measured amounts are superphy iologic amount ⁇ . While not wi ⁇ hing to be bound by any particular theory of action or operation, the inventor ⁇ believe that administration of doses containing superphysiologic amounts of hormonally ineffective insulin related polypeptides on a therapeutic T cell modulatory schedule in accordance with their invention results in particularly beneficial effects on the development and severity of type 1 diabetes.
  • Doses containing superphysiologic amounts of a hormonally ineffective insulin related polypeptide mean ⁇ do ⁇ e ⁇ providing amounts of a hormonally ineffective insulin related polypeptide, or of a combination of hormonally ineffective insulin related polypeptides, that would cause insulin ⁇ hock if the in ⁇ ulin related polypeptide( ⁇ ) were hormonally effective.
  • a superphy ⁇ iologic amount contains a number of moles of the hormonally ineffective in ⁇ ulin related polypeptide equal to or greater than the number of moles of hormonally effective insulin that will induce insulin shock in the patient.
  • the hormonally ineffective insulin related polypeptide is preferably ⁇ elected from the group con ⁇ i ⁇ ting of pre-pro insulin, proin ⁇ ulin, denatured in ⁇ ulin, in ⁇ ulin chain B, in ⁇ ulin chain A, and in ⁇ ulin chain C, either a ⁇ whole polypeptides or fragments of these polypeptides. Combinations of these polypeptides may also be administered in accordance with the present invention as long as their combination does not generate a hormonally effective insulin polypeptide. Particular ⁇ of ⁇ uch hormonally ineffective in ⁇ ulin related polypeptides are discussed below under the heading "Detailed Description of the Preferred Embodiments.”
  • the hormonally ineffective in ⁇ ulin related polypeptide (or polypeptides) used in the practice of the present invention is preferably a form or fragment of an insulin molecule comprising an amino acid ⁇ equence of at lea ⁇ t 6, and preferably 10 con ⁇ ecutive amino acid ⁇ that are in a ⁇ equence identical to that of the ⁇ equence of an insulin molecule that is hormonally effective. More preferably the hormonally ineffective insulin polypeptide is selected from the group con ⁇ isting of pre-pro insulin, proinsulin, denatured in ⁇ ulin, insulin chain B, insulin chain A, and insulin chain C, either as whole polypeptides or as fragments of these polypeptides.
  • a particularly preferred hormonally ineffective insulin related polypeptide for use in the present invention comprises insulin Chain B. Most preferred is in ⁇ ulin Chain B that i ⁇ ⁇ ubstantially free of other insulin chains.
  • hormone ineffective refer ⁇ to hormonal effectivene ⁇ s in a patient, preferably a human patient, although such effectivene ⁇ i ⁇ conveniently mea ⁇ ured u ⁇ ing the rabbit blood- ⁇ ugar method (USP biological te ⁇ t ⁇ 121>, USP 23 / NF 18 1995) .
  • suitable assays of the hormonal activity of insulin are known in the art, and may be used if desired to determine the level of hormonal effectiveness or ineffectivenes ⁇ of a particular preparation of one or more in ⁇ ulin related polypeptide ⁇ .
  • Hormonally ineffective in ⁇ ulin polypeptide ⁇ ⁇ uitable for use in the practice of the present invention can be prepared by conventional mean ⁇ well known in the art, including: purification from natural ⁇ ource ⁇ (i.e., from animal pancreases, e.g.
  • hormonally effective insulin ⁇ may be prepared by any of the various means known in the art, and may then be inactivated, preferably by denaturation (followed by chain separation, if desired) .
  • Denaturation of hormonally effective insulin ⁇ to produce hormonally ineffective insulin polypeptides may be carried out by any method of denaturation, a large variety of which are known to those of skill in the art. These include heat denaturation, various types of chemical denaturation (e.g., using chaotropic agents such as urea or guanidinium HCl, or other chemical means of denaturation such as carboxymethylation) . Denaturation of in ⁇ ulin related polypeptide ⁇ produced by ⁇ ynthetic or recombinant DNA method ⁇ may al ⁇ o be achieved by ⁇ equence truncation or in ⁇ ulin precursor ⁇ ynthesi ⁇ in organi ⁇ m ⁇ incapable of proce ⁇ ing the precursor to produce mature, hormonally effective insulin.
  • combinations of these hormonally ineffective insulin related polypeptides may also be u ⁇ ed in the practice of the pre ⁇ ent invention as long as their combination does not generate a hormonally effective in ⁇ ulin polypeptide.
  • a ⁇ used herein a hormonally ineffective insulin related polypeptide has le ⁇ hormonal activity than the minimum activity for insulin required by the United States Pharmacopea (USP 23 / NF 18, 1995) , which is 26 USP units per milligram. Therefore, in accordance with the present invention, any insulin related polypeptide exhibiting 25 USP unit ⁇ of in ⁇ ulin activity per milligram or le ⁇ i ⁇ considered to by hormonally ineffective.
  • the hormonally ineffective insulin related polypeptide has les ⁇ than 20 USP unit ⁇ of activity per mg, more preferably less than 10 USP units per mg, and most preferably les ⁇ than 5 USP unit ⁇ of in ⁇ ulin activity per mg of polypeptide.
  • doses containing superphysiologic amounts of hormonally ineffective insulin related polypeptide ⁇ mean ⁇ doses containing amounts providing at least an equivalent number of mole ⁇ of hormonally ineffective insulin related polypeptide to the number of moles of hormonally effective insulin that will induce insulin ⁇ hock in the patient.
  • such superphy ⁇ iologic amounts of hormonally ineffective insulin related polypeptide ⁇ compri ⁇ e amounts ranging from about 6.9 pM/kg/patient to about 8.6 ⁇ M/kg/patient.
  • the amounts range from about 34.5 pM/kg/patient to about 5.2 ⁇ M/kg/patient. More preferably the amounts range from about 170 pM/kg/patient to about 3.5 ⁇ M/kg/patient.
  • Most preferably the amounts range from about 0.5 ⁇ M/kg/patient to about 3.5 ⁇ M/kg/patient.
  • the present invention also provides for the repeated adjuvant free administration of doses containing lower (not superphy ⁇ iologic) amounts of hormonally ineffective insulin related polypeptides to a patient in need of ⁇ uch treatment.
  • doses containing amounts of below about 6.9 pM/kg/patient, and as low as about 1 pM/kg/patient may be u ⁇ ed in the practice of the pre ⁇ ent invention.
  • ⁇ uch lower amounts are referred to as "physiologic doses" or "physiologic amounts” when they contain a number of moles of hormonally ineffective insulin related polypeptide that is less than the number of moles of hormonally effective insulin that will induce insulin shock in the patient, but contain at least about 1 picomole of hormonally ineffective insulin related polypeptide, i.e., at least as many mole ⁇ of in ⁇ ulin related polypeptide as there are moles of insulin in about 0.15 USP units of insulin with a specific activity equal to the USP minimum activity standard of 26 units per mg (the equivalent of about 5.8 ug of human insulin with that specific activity) .
  • a therapeutic T cell modulatory schedule involves administration of doses containing superphy ⁇ iologic amounts of the hormonally ineffective in ⁇ ulin polypeptide ⁇ repeatedly to the patient at least two times at an interval of at least six, and preferably at least twelve hours and not more than seven days between doses.
  • the polypeptides are administered parenterally without the concomitant administration of an adjuvant.
  • Administration by a parenteral route will typically be via injection such as intravascular injection (e.g., intravenous infusion), subcutaneous injection, or intramuscular injection.
  • Other non- oral routes of administration e.g., mucosal, inhalation, transdermal ultrasound, and the like, may be used if desired and practicable for the particular hormonally ineffective insulin related polypeptide to be administered.
  • Formulations suitable for injection and other routes of administration are well known in the art and may be found, for example, in Remington's Pharmaceutical Sciences. Mack Publi ⁇ hing Company, Philadelphia, PA, 17th ed. (1985) .
  • Preferred formulations for parenteral administration of hormonally ineffective insulin related polypeptides are those described for insulin in the USP 23/NF 18 (1995) .
  • Parenteral formulations mu ⁇ t be ⁇ terile and non-pyrogenic, and generally will include a pharmaceutically effective carrier, ⁇ uch a ⁇ saline, buffered (e.g., phosphate buffered) saline, Hank' ⁇ ⁇ olution, Ringer's solution, dextrose/saline, glucose solutions, and the like.
  • Formulations may al ⁇ o contain pharmaceutically acceptable auxiliary ⁇ ub ⁇ tances as required, such as, tonicity adjusting agent ⁇ , wetting agent ⁇ , bactericidal agents, preservatives, stabilizers, and the like.
  • Non-obese diabetic mice are a strain of mice that are prone to the development of diabetes, and repre ⁇ ent an accepted model system for the study of diabetes mellitus.
  • NOD SCID mice de ⁇ ignated NOD/SCID, are available from the Jack ⁇ on Laboratorie ⁇ , Bar Harbor, ME. The incidence of diabete ⁇ by 200 day ⁇ of age in these mice is 80% in females and 50% in males. At 110 days of age, fewer than 15% of the male NOD mice became diabetic.
  • CYTOXAN Cyclophosphamide
  • CYTOXAN Cyclophosphamide
  • mice All the mice were subjected to urine gluco ⁇ e mea ⁇ urement 2- 3 times per week for a period of 21 days after cyclophosphamide injection. The onset of diabetes was recorded as the first point at which two consecutive days of po ⁇ itive urine glucose result ⁇ were obtained. Blood glucose was also measured to confirm the urine glucose re ⁇ ults. (ExacTech, MediSense Inc., Cambridge, MA) In all the mice tested with po ⁇ itive urine gluco ⁇ e, blood gluco ⁇ e level ⁇ were greater than 150mg/dl. At day 21, the ⁇ e mice were ⁇ acrificed and examined hi ⁇ tologically.
  • Oxidized bovine insulin Chain B was purchased from Sigma (catalog no. 1-6383) . Both insulin chain B and the BSA control protein (Miles Inc., #81-001) were dissolved in PBS/1M HCL, pH2, before dialysi ⁇ against PBS, pH7.2, sterile filtration, and storage of frozen aliquots. Treatment;
  • In ⁇ ulin Chain B treatment in the CYTOXAN model Groups of randomly selected NOD mice were injected intravenously twice daily with either 500ug insulin Chain B or 500ug BSA on day ⁇ 1, 3, and 5 following CYTOXAN treatment (day 0) .
  • In ⁇ ulin Chain B treatment in the adoptive tran ⁇ fer model NOD/SCID mice were injected intravenously twice daily with 500ug of in ⁇ ulin Chain B (or BSA as control) on day 3, 5 and 7 relative to the time of spleen cell transfer (day 0) .
  • Table-1 do ⁇ e dependency of cyclophosphamide induced diabetes in non-diabetic male NOD recipients

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Abstract

Novel immunomodulatory methods for both the prevention and treatment of type I diabetes mellitus are provided. These methods comprise the administration of doses containing measured amounts (preferably superphysiologic amounts) of an adjuvant free hormonally ineffective insulin related polypeptide to the patient. Preferably this administration is carried out on a schedule designed to induce apoptosis, anergy, or other modulation of the autoimmune activity of T cells reactive with at least one epitope of the hormonally ineffective insulin related polypeptide. The hormonally ineffective insulin related polypeptide is preferably selected from the group consisting of pre-pro insulin, proinsulin, denatured insulin, insulin chain B, insulin chain A, and insulin chain C, either as whole polypeptides or fragments of these polypeptides. Combinations of these polypeptides may also be administered as long as their combination does not generate a hormonally effective insulin polypeptide. The administration of hormonally ineffective insulin related polypeptides in accordance with the invention results in beneficial effects on the development and severity of type I diabetes.

Description

TREATMENT OF DIABETES VIA ADMINISTRATION OF HORMONALLY
INEFFECTIVE INSULIN
BACKGROUND OF THE INVENTION
The discussion in this section is not limited to subject matter that qualifies as "prior art" against the present invention. Therefore, no admission of such prior art status shall be implied or inferred by reason of inclusion of particular subject matter in this discussion, and no declaration against the present inventors' interests shall be implied by reason of such inclusion.
The teachings and disclosures, in their entireties, of the following documents are hereby incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains;
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Diabetes Mellitus Diabetes mellitus is the most common endocrine disease, and is characterized by abnormalities of glucose metabolism. The abnormal glucose metabolism associated with this disease results in hyperglycemia (high blood glucose levels) and eventually causes complications of multiple organ systems, including eyes, kidneys, nerves, and blood vessels. Patients with persistent hyperglycemia or abnormal glucose tolerance are generally diagnosed with the disease, although most commonly patients initially present with excessive urination (polyuria) and frequent drinking due to extreme thirst (polydipsia) . These typical initial symptoms result from the osmotic effects of hyperglycemia.
The pathogenesis of diabetes mellitus is typically associated with pancreatic dysfunction, particularly of the beta cells of the pancreatic islets of Langerhans. This dysfunction may lead to destruction of the islet beta cells, which produce insulin, a glucose regulatory peptide hormone. Diabetes mellitus has been generally categorized as insulin dependent or type 1, versus non-insulin dependent, or type 2. However, this terminology has evolved as the disease has become better understood. For example, it has been found that in some patients suffering from non-insulin dependent diabetes, the disease progresses into an insulin dependent form, while in other patients insulin dependence does not develop.
Patients are thus often categorized in terms of the mechanisms of pathogenesis of islet destruction, and the designation type 1 is now used to refer to autoimmune islet pathogenesis, i.e., to diabetes caused by islet-specific autoimmune attack, and is εo used hereinafter. The term insulin dependent diabetes mellitus (IDDM) refers to type 1 diabetes that has progressed to a stage where enough autoimmune destruction of the pancreatic beta cells has occurred to produce overt symptoms. The term pre-IDDM refers to an autoimmune condition that can be detected by biopsy or by analysis of autoimmune responses, in which pancreatic islet beta cells are being subject to a specific autoimmune attack to an extent where some cells may be subject to destruction. In pre-IDDM, however, the destruction (if any) has not progressed to an extent sufficient to require the administration of insulin. Since there can be a point in the early stages of type 1 diabetes in which overt symptoms are observed but some islet function remains, not all type 1 diabetes is classified as IDDM, and not all pre-IDDM presents without overt symptoms. Complications of Type 1 Diabetes
The metabolic complications associated with the abnormal metabolism caused by insulin insufficiency can affect numerous organ systems. The most common acute metabolic complication is that of diabetic ketoacidosis, characterized by severe hyperglycemia (and resulting hypovolemia caused by osmotic diuresis) as well as metabolic acidosis induced by excess free fatty acid release and the production of ketone bodies. A patient experiencing these complications may present with anorexia, nausea, vomiting and/or altered consciousness, and must be treated aggressively with insulin therapy as well as fluid and electrolyte replacement.
In addition to the acute metabolic complication of ketoacidosis, the diabetic patient is susceptible to a series of late complications that cause considerable morbidity and premature mortality. Atherosclerosis occurs more extensively and earlier in diabetics than in the general population as a result of abnormalities in both glucose and lipid metabolism. This vascular pathology can lead to, inter alia, coronary artery disease, stroke, and peripheral vascular disease with gangrene. Retinopathy iε another vascular complication of diabetes. Diabetic retinopathy is a leading cause of blindness, and is initiated by increased permeability of retinal capillaries which can progress to occlusion, hemorrhage, aneurysm formation, and neovascularization known as proliferative retinopathy.
In addition to vascular complications, kidney and neurological diseases (nephropathies and neuropathieε) are common complications of diabetes. Diabetic nephropathy causes about half of end-stage renal disease in the United States. Histologically, the nephropathy is characterized by glomerular basement membrane widening and mesangial thickening. Initial signs include increasing proteinuria, with azotemia ultimately leading to renal failure. Diabetic neuropathy can affect any part of the nervous system, with the possible exception of the brain. The neuropathy is most commonly seen aε peripheral polyneuropathy, with symptoms including numbness, paresthesias, severe hyperesthesias, and pain. Autonomic neuropathy can cause gastrointestinal dysfunction, orthostatic hypotension, bladder dysfunction or paralysis, and impotence. Diabetic foot ulcers represent a special problem of diabetics, and appear to be due primarily to abnormal pressure distribution secondary to diabetic neuropathy. The ulcerous lesions are often worsened by concomitant peripheral vascular disease and infection. As mentioned above, meticulous control of blood glucose has been associated with amelioration of the late complications of diabeteε, εuggesting that therapieε that preserve beta cell function could reduce or eliminate the majority of the complications of diabetes mellitus. Pathogenesis of Tvpe 1 Diabetes
Type 1 diabetes only develops in genetically susceptible individuals, and symptoms generally appear before age 40, with the peak incidence of onset of overt symptomology occurring in the second decade of life. The pathogenesis of type 1 diabetes is characterized by an initial phase of leukocyteε infiltration into the islets, referred to aε inεulitiε, followed over a period of time by the actual deεtruction of the islet beta cells by autoimmune attack. The insulitis phase is characterized by infiltration of pancreatic islets by both lymphocytes and cells of the monocyte/macrophage lineage, and entailε both cell- mediated inflammation as well aε attack by islet-specific cytotoxic antibodies. Overt clinical symptoms of diabetes mellitus are generally manifested when over 90% of the iεlet beta cells are destroyed, however, as discussed more fully below, it is now poεεible to accurately detect individualε undergoing earlier stages of type 1 pathogenesis, i.e., before enough islet beta cells have been lost to produce overt clinical symptoms.
The autoimmune process is generally thought to be induced by an environmental stimulus. One reason for thiε belief iε that an identical twin haε only a fifty / fifty chance of developing IDDM if hiε identical εibling haε the diεeaεe. T Cells The autoimmune destruction of the beta cells of the pancreatic islets in Type 1 diabetes is believed to be initiated by white blood cells (leukocytes), moεt importantly T cellε. T cells, or T-lymphocytes, are mononuclear white blood cells that provide many essential immune functions. The importance of T cells in human autoimmune diseases has been increasingly appreciated in the past decade. Studies using treatments that result in generalized immunoεuppression have defined a critical role for a subset of T cellε, known aε CD4+ or helper T cellε, aε primary regulators of all immune reεponses (both cellular and humoral) to protein or peptide antigenε.
T cells mediate tissue injury by indirect and direct means.
T cellε of both CD8+ (cytotoxic) and CD4+ (helper) εubsetε secrete a variety of inflammatory cytokines that can damage tissueε indirectly by activating various other types of white blood cellε. Exampleε of εuch T cell effects include activation of antibody εecreting B cellε (εtimulating humoral immune activity) and activation of macrophageε, which can cauεe acute tiεsue damage and inflammation by releasing hydrolytic enzymes, reactive oxygen species, and additional pro-inflammatory cytokines. In addition to theεe indirect effectε of T cell activity, direct tiεεue damage can be mediated by CD8+ cytotoxic T cells attacking cells displaying target antigenε.
One unique aεpect of the phyεiology of T cellε iε the presence of membrane bound antibody-like binding structures called T cell receptors (TCRs) on their cell εurfaceε. Like antibodieε, TCRε bind with high specificity to particular antigens. Like antibody-producing cellε, which develop as multitudinous clones of cells, each clone producing antibodies with unique specificities, T cells develop as a vast number of distinct clones, and any particular T cell clone expresses a single type of TCR with a defined binding specificity. T cell clones with TCRs that bind specifically to self antigens are responεible for the development of autoimmune diεeases.
Studies of the interactions of antibodies and TCRs with their specific antigens have shown that a particular polypeptide antigen typically comprises numerous εubmolecular features, known as epitopes, that each can serve aε a diεtinct binding site for a particular antibody or TCR. T Cells and Autoimmune Diseases
In autoimmune diseases, only a limited number of T cell clones, reactive with various epitopes of a small number of autoantigens, become activated and are involved in pathogenesis. Even in individuals suffering from autoimmune diseases, only a small subset of T cell clones (0.1-1%) are known to recognize autoantigens.
Variouε mechanismε have been poεtulated to play a role in the pathogenic activation of diseaεe-cauεing autoreactive T cellε. Primary activation of antigen presenting cells (APCs) by infection or local inflammation is implicated in one such mechanism. APCs activated in this way can then provide powerful co-stimulation for hitherto unreactive T cells.
Other proposed mechanisms involve the polyclonal activation of previously quiescent autoreactive T cells by superantigens, such as bacterial toxins; or a coincidental molecular mimicry between foreign and self antigens (Abbas et. al. 1994) . In this last case, the host immune system mounts a response to an epitope on a protein expressed by a pathogen, such as a virus, that resembles a homologous epitope on a host protein. Autoimmune attack then reεultε from the cross-reactive immune responεe that ensues.
In addition to external factors, underlying the emergence of all T cell-mediated autoimmune disease is a complex pattern of inherited susceptibility determined by multigenic factors. For further diεcusεions of these various factors, Steinman, 1995, reviews current theories of autoimmunity.
Alterations in the T cell repertoire occur naturally during T cell development. Only a small fraction of thymocytes (immature T cells) survive the intrathymic development and selection events that result in emigration of developing T cells to the peripheral circulation and the completion of their maturation (von Boehmer, 1988; Marrack and Kappler, 1987) . Experimental evidence strongly εuggeεtε that a large number of thymocytes that bear receptors for autoantigenε are initially present in the thymus. Recent studies have yielded evidence suggesting that a procesε referred to aε programmed cell death, or apoptosis, destroys these autoreactive thymocytes in the thymus while sparing thymocytes that are not autoreactive. Apoptosiε thuε playε a large role in shaping and maintaining the T cell repertoire and contributes to the establishment of self- tolerance by actively eliminating cells expressing autoreactive TCRs.
It has recently been discovered that T cells are sensitive to apoptotic cell death induced by a variety of stimuli at multiple points in their lifeεpan (see, for example, Lenardo 1991; Boehme and Lenardo 1993; Critchfield et al. 1994) . Positive selection factors are also believed to play a role in regulating the survival of specific T cell clones. The reduction or expansion of the number of individual T cells of a particular clone in an organism by these and other mechanisms serve to modulate the responsivenesε of the organiεm's immune syεtem to a particular antigen. It iε now firmly eεtabliεhed in several autoimmune diεeaεe modelε, aε well aε in certain viral infectionε, that apoptosis can be induced (upon exposure to antigen under certain defined conditionε) in mature peripheral antigen-εpecific T lymphocyteε aε well aε in immature thymocyteε.
Apoptosis occurs in many biological syεtemε (see, for example, Kerr et al. 1991; Lockεhin and Zakeri, 1991; Cohen et al. 1992; Duvall and Wyllie, 1986; Cotter et al. 1990) . A cell undergoing apoptoεiε undergoeε a εpecific program of eventε -- cellular and biochemical proceεεeε that depend upon active metaboliεm and contribute to the cell's self-deεtruction. In apoptotic T cellε, the nucleus shrinks, the chromatin condenses, the genetic material (DNA) progressively degrades into εmall (nucleoεomal repeat εized) fragmentε, there iε cytoplaεmic compaction, the cell membrane forms blebs, and the cell eventually collapses (Kawabe and Ochi, 1991; Smith et al. 1989) . Cells cannot recover from apoptosis, it results in irreversible cell death (Kawabe and Ochi, 1991; Smith et al. 1989) . Recent reports have εuggeεted a role for the TNF-related cytokine known as the FAS ligand and its receptor, CD95 (the FAS receptor) , in the induction of apoptosiε in T cells (Crispe et al. 1994; Nagata and Suda, 1995; Straεεer, 1995; Dhein et al. , 1995; Brunner et al. , 1995; and Ju et al. , 1995) . T cells that do not undergo apoptosis, but which have become activated, will carry out their "effector" functions by causing cytolysis, or by secreting lymphokineε that cauεe B cell responseε or other immune effectε (Paul, 1989) . These effector functions are the cause of tissue damage in autoimmune and other diseases.
Islet Beta Cell Autoantigens
As discussed above, the onset of Type 1 diabetes is conεidered to be mediated by T cellε. The diεease is believed to be a consequence of inappropriate εpecific T cell responses to certain islet beta cell proteins that act as autoantigens. In addition to autoreactive T cells, autoantibodies against variouε self antigens have also been reported in IDDM patients. The antigens reported to be bound by these autoantibodieε include many of those that have been reported to be recognized by autoreactive T cells.
Autoantigens that are subject to autoimmune responεes in Type 1 patients include εialyglycolipid; the 64kDa and 67kDa GAD (glutamate decarboxylaεe) autoantigenε; insulin; a 38kD antigen from the secretory granuleε of beta cells; an antigen crosε reactive with antibodies to bovine albumin known aε the beta cell p69 protein, PM-1, or diεease-modifying antigen, a beta cell cytoεkeletal protein known as peripherin, glucoεe tranεporter proteins, including GLUT-2 ,* heat shock protein 65 (HSP 65) , including the p277 peptide; carboxypeptidaεe H; a 52Kd molecular mimic of Rubella viruε antigen; a beta cell membrane aεsociated protein of 150kDa; a protein antigen located at the secretory pole of the rat insulinoma cell line RINm38, referred to aε the RIN polar antigen; and poorly characterized antigenε isolated by immunoscreening of an islet cDNA expreεεion library, referred to as ICA12 and ICA512. The relative importance of these various autoantigens to autoimmune pathogenesiε, and the timing with which each plays a role during the course of disease onset and development, are the subject of considerable uncertainty and consequent controversy in the art. Further uncertainty stems from the fact that each supposed autoantigen compriseε numerous epitopes, some of which may be have disease promoting effects while others may have disease suppreεεive effectε.
Autoantibodieε to 64kD GAD normally are detected before the onset of clinical insulin dependent type 1 diabetes mellitus, and among nondiabetic relatives of patientε with IDDM aε well aε otherε at riεk. These autoantibodies have been suggested to be the best predictive autoantibody marker for impending diεease. There are two formε of GAD proteinε, GAD 65 and GAD 67, encoded by different geneε on different chromosomes, that are about 70% homologous. Human iεletε only express GAD 65, although both protein forms are found in the brain. Evidence of lymphocyte specific immunity to GAD has been demonstrated and found to be closely asεociated with IDDM. Recent studies in the NOD mouse model of diabetes haε indicated that T cell reεponses to GAD 65 precede those to other putative autoantigenε and that early induction of T cell tolerance to GAD can prevent onset of disease.
Kaufman et al (1993) and Tisch et al (1993) have presented data that εuggest that GAD responses are the most important in diseaεe development, aε they were reported to ariεe firεt during the development of type 1 diabeteε, with responseε to other beta cell autoantigenε only appearing much later in the course of the diseaεe, with inεulin reactivity being amongεt the last to appear. These findings were interpreted as indicating that GAD is the key autoantigen in type 1 diabetes, and that modulation of autoimmune reactivity with GAD would be the most appropriate target for reducing diεeaεe pathology.
In accordance with this theoretical understanding of disease progression, modulation of insulin reactive T cells would be closing the barn door after the horses had gone, the anti-insulin reactions being observed so late in disease progression that their modulation would not be expected to affect the onset or severity of diεeaεe.
Responseε of T cells from type 1 diabetes patients or at- riεk individualε to undefined islet cell preparations have suggested that T cells also respond to other islet cell antigens. Theεe include a 38kD antigen from the εecretory granuleε of beta cells, and serum albumin. In addition, heat shock protein 65 has been implicated based upon the fact that HSP-specific T cells were shown to transfer disease in NOD mice. Carboxypeptidase H iε a molecule found in islet secretory granules and is associated with the production of peptide hormoneε and neurotranεmitterε. It was identified as a potential islet autoantigen by the screening of cDNA expression libraries with sera from IDDM or pre-IDDM patientε. Several other putative iεlet cell antigenε, such as ICA12 and ICA512, have also been identified by screening of cDNA expression libraries.
Insulin autoantibodies (IAA) can be detected in around 50% of new onset patients, and are highly associated with islet cell autoantibodies (ICA) and the HLA-DR4 phenotype. Other studieε εuggeεt that individualε with both ICA and IAA have a much higher riεk for developing overt type 1 diabeteε than those with either marker alone. T cell responses to insulin as autoantigen have alεo been deεcribed. In one study cellular responses to human insulin were present in almost 90% of ICA-positive first degree relatives of IDDM patients. Also, as discussed below in the examples, insulin reactive T cells from diabetic NOD mice can tranεfer diabetes to non-diabetic NOD mice. Prediction of Type 1 Disease As discusεed above, there iε a genetic aspect to the incidence of type 1 diabetes. Accordingly, individuals with a known family history of the diεeaεe can be monitored for early, preclinical εigns of disease development, e.g., by monitoring levels of the autoantibodies discuεsed herein. In addition, genetic tests can identify certain individuals at increased riεk of developing the disease (see, for example, Walston et al . 1995) .
Among the autoantibodies known to be asεociated with type 1 diabetes, those directed against GAD are the ones that appear earliest and are present in the largest number of patients. Overall, recent studies have shown that over 80% of individuals with preclinical diabetes have GAD-specific autoantibodies. In thiε case an individual with preclinical diεease is defined as a first degree relative of a type 1 diabetes patient with ICA. The antigens identified by ICA are ill-defined, but together with IAA and GAD-specific autoantibodies, they yield a high predictive value for onset of diabeteε in preclinical individuals. Interestingly, in actual early onεet diseaεe, the frequency of GAD-specific antibodies declineε. This could be due to the fact that GAD reactivity declines with beta cell destruction.
Prediction of type 1 diabetes may also be facilitated by monitoring of the subject's blood sugar levels, preferably, in conjunction with the administration of a glucose tolerance test to the subject. Such procedures are preferably carried out in combination with the monitoring of titers of the subject's circulating autoantibodies, such autoantibodies selected from the group consiεting of IAA, ICA, and GAD εpecific autoantibodieε.
Current Methods for Prevention and Treatment of Type 1 Diabetes.
While diabeteε has been studied for centuries, only a few effective treatments are available for type 1 diseaεe. The first line of treatment is diet, with appropriate caloric intake based on ideal body weight and a defined distribution among protein, glucose, and fat. However, in IDDM patients, the most important component of therapy is the administration of insulin, the goal of which is to maintain glucose levels as cloεe to the normal range aε possible throughout the day. Insulin is available in rapid, intermediate, and long-acting formulations which vary in onset, peak, and duration of action, and can be uεed in varying εcheduleε of adminiεtration to attempt to optimally regulate plasma glucose levels.
Intensive insulin therapy referε to a rigorouε regimen of adminiεtration of hormonally effective inεulin and monitoring of blood εugar levels. Thiε regimen is designed to control blood glucoεe as precisely as posεible. The reεultε of the multicenter Diabetes Control and Complication Trial established that complications of diabeteε are εignificantly diminished by better control of blood glucose levelε, and thuε demonεtrated the desirability of intensive insulin therapy. One problem with this approach iε that intensive insulin therapy requires a high level of patient awareness and compliance, aε well as a highly skilled care team of physicians, nurses, and dietitians. The goals of intensive insulin therapy are thus extremely difficult to achieve, even with motivated and educated patients. Another problem is that a higher rate of hypoglycemia is seen in such rigorously treated patients than in patientε receiving standard, less rigorous, inεulin regimenε.
The Diabetes Control and Complication Trial highlighted not only the benefit to overall metabolic health of maintaining normal blood glucose levels, but alεo a fundamental problem aεεociated with the treatment of type 1 diabetes, namely that the overt symptoms of the disease are manifested only when essentially all of the patients' islets are destroyed. Oral agents for diabetes, such aε the sulfonylureas, act primarily by stimulating the release of insulin from dysfunctional beta cells, and thus are not useful for most patients with type 1 disease, i.e. for those patients with IDDM.
A major unmet goal in the treatment of diabeteε has been to develop a therapy capable of aborting the autoimmune attack on the islet beta cells prior to their complete destruction, thereby preserving enough endogenous function to maintain normal metabolic control. Induction of tolerance. In the NOD (non-obeεe diabetic) mouεe model of diabeteε, it has been shown that oral feeding of insulin delayed the onset and reduced the severity of the disease. The mechanism proposed to explain oral tolerance is that oral antigen administration induceε populations of antigen-specific Th2 T cells that secrete antiinflammatory cytokines such as IL-4, IL-10, and TGF-beta.
These T cells circulate and are activated to secrete cytokines only in the presence of their εpecific antigen. Thuε, inεulin- specific Th2 T cells would be activated only in the pancreas where they would produce εuppreεεive cytokines to modulate the autoimmune process. This mechanism does not require, therefore, that the oral antigen actually represent a diseaεe-specific autoantigen, but rather only that it is expressed in a tisεue specific fashion.
In contrast, methodε deεigned to produce T cell tolerance (e.g., by anergy or apoptoεiε) require the identification of the actual diseaεe-specific autoantigens that are targeted by autoimmune attack. Such antigens are then adminiεtered to patientε in an appropriate tolerizing fashion (which may alεo induce non-antigen-specific tolerizing effects) . Given that type 1 diabetes is in significant measure a diseaεe mediated by islet-specific autoreactive T cells, therapy of this type εhould be feaεible in principle. Thuε, induction of neonatal tolerance to GAD, aε referred to above, prevented onεet of diεeaεe in NOD mice. In addition, injection of crude iεlet extractε intrathymically, where tolerization of developing T cellε takeε place, haε alεo protected both NOD mice as well aε pre-diabetic BB rats from developing clinical disease. One approach taken to induce insulin tolerance involves the parenteral administration of insulin, in combination with a conventional adjuvant (Freund's adjuvant), of low doseε (doεes below those expected to cause insulin shock in the patient if an equimolar dose of hormonally effective insulin were administered to the patient) of insulin Chain B or Chain A, or of intact, hormonally effective insulin. Apoptosiε
Apoptoεiε is a form of programmed cell death that occurs in many biological systems (Kerr et al. , 1991. Apoptosis: The molecular basiε of cell death, Tomei and Cope (eds.), Cold Spring Harbor Laboratory Press, Plainview, New York, pp. 5; Lockεhin and Zakeri, 1991. supra, pp. 47; Cohen et al . , 1992. Ann Rev Immunol 10, pp. 267; Duvall and Wyllie, 1986. Immunol Today 7, pp. 115; Cotter et al. , 1990. Anticancer Research 10, pp. 1153) . An apoptotic cell undergoes a εpecific program of eventε that depend upon active metabolism and contribute to its own self-destruction. In apoptotic T cells, the nucleus shrinks, the genetic material (DNA) progresεively degradeε, and the cell eventually collapεeε (Kawabe and Ochi, 1991. Nature 349, pp. 245-248; Smith et al. , 1989. Nature 337, pp. 181-184) . Cellε cannot recover from apoptoεis, it resultε in irreverεible killing (Kawabe and Ochi, 1991. Nature 349, pp. 245-248; Smith et al., 1989. Nature 337, pp. 181-184) . T cellε that do not undergo apoptoεiε, but which have become activated, will carry out their "effector" functionε by cauεing cytolyεiε, or by εecreting lymphokines that cauεe B cell responses or other immune effects (Paul, 1989. Fundamental Immunology, 2nd Ed. Paul (ed.), Raven Presε, New York, pp. 3-38) . These "effector" functions are the cauεe of tiεεue damage in autoimmune and other diεeaseε. A powerful approach to avoiding disease is thus to permanently eliminate by apoptosis only those T cells reactive with autoimmune disease-inciting antigens, while leaving the majority of the T cell repertoire intact. The use of auto¬ antigens to carry out this approach is described in PCT patent publication No. 94/28926, filed in the name of Michael J. Lenardo, and entitled Interleukin-2 Stimulated T Lymphocyte Cell Death for the Treatment of Autoimmune Diseases, Allergic Disorders, and Graft Rejection, and PCT patent publication No. 94/03202, filed in the name of Michael J. Lenardo, Stefen A. Boehme, and Jeffrey Critchfield and entitled Interleukin-4 Stimulated T Lymphocyte Cell Death, both of which patent publications are incorporated herein by reference. The accompanying figures, which are incorporated in and constitute part of the specification, illustrate certain aspectε of the invention, and together with the deεcription, εerve to explain the principles of the invention. It is to be understood, of course, that both the figures and the description are explanatory only and are not restrictive of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of the CYTOXAN induced IDDM experiments . Figure 2 showε the reεultε of the adoptive tranεfer of IDDM experimentε. SUMMARY OF THE INVENTION
In view of the foregoing, the objects of this invention are to provide novel immunomodulatory methods for both the prevention and treatment of type 1 diabetes mellitus. To achieve this and other objects, the invention provides a method of treating a patient in need of such treatment, e.g., a patient selected from the group of patients consisting of patients at risk of developing diabetes and patients suffering from diabetes (particularly early in disease progression before all of the patient's beta cells have been destroyed by the disease), so as to delay the onset or reduce the symptoms of diabetes in the patient. This method comprises the administration of doεeε containing meaεured amounts of an adjuvant free hormonally ineffective insulin related polypeptide to the patient. Preferably thiε adminiεtration is carried out on a therapeutic T cell modulatory schedule, i.e., a schedule designed to induce apoptosis, anergy, or other modulation of the autoimmune activity of T cells reactive with at least one epitope of the hormonally ineffective insulin related polypeptide. Particulars of such therapeutic T cell modulatory scheduleε are diεcussed below under the heading "Detailed Description of the Preferred Embodiments."
Preferably the measured amounts are superphy iologic amountε. While not wiεhing to be bound by any particular theory of action or operation, the inventorε believe that administration of doses containing superphysiologic amounts of hormonally ineffective insulin related polypeptides on a therapeutic T cell modulatory schedule in accordance with their invention results in particularly beneficial effects on the development and severity of type 1 diabetes.
Doses containing superphysiologic amounts of a hormonally ineffective insulin related polypeptide meanε doεeε providing amounts of a hormonally ineffective insulin related polypeptide, or of a combination of hormonally ineffective insulin related polypeptides, that would cause insulin εhock if the inεulin related polypeptide(ε) were hormonally effective. In accordance with the preεent invention, such a superphyεiologic amount contains a number of moles of the hormonally ineffective inεulin related polypeptide equal to or greater than the number of moles of hormonally effective insulin that will induce insulin shock in the patient. Particulars of such doses and amounts are discusεed below under the heading "Detailed Deεcription of the Preferred Embodiments. "
The hormonally ineffective insulin related polypeptide is preferably εelected from the group conεiεting of pre-pro insulin, proinεulin, denatured inεulin, inεulin chain B, inεulin chain A, and inεulin chain C, either aε whole polypeptides or fragments of these polypeptides. Combinations of these polypeptides may also be administered in accordance with the present invention as long as their combination does not generate a hormonally effective insulin polypeptide. Particularε of εuch hormonally ineffective inεulin related polypeptides are discussed below under the heading "Detailed Description of the Preferred Embodiments."
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hormonally Ineffective Insulin Polypeptides
The hormonally ineffective inεulin related polypeptide (or polypeptides) used in the practice of the present invention is preferably a form or fragment of an insulin molecule comprising an amino acid εequence of at leaεt 6, and preferably 10 conεecutive amino acidε that are in a εequence identical to that of the εequence of an insulin molecule that is hormonally effective. More preferably the hormonally ineffective insulin polypeptide is selected from the group conεisting of pre-pro insulin, proinsulin, denatured inεulin, insulin chain B, insulin chain A, and insulin chain C, either as whole polypeptides or as fragments of these polypeptides. A particularly preferred hormonally ineffective insulin related polypeptide for use in the present invention comprises insulin Chain B. Most preferred is inεulin Chain B that iε εubstantially free of other insulin chains.
Aε uεed herein, the hormonal effectiveness, or lack thereof, indicated by the phraseε "hormonally effective" and
"hormonally ineffective" referε to hormonal effectiveneεs in a patient, preferably a human patient, although such effectiveneεε iε conveniently meaεured uεing the rabbit blood-εugar method (USP biological teεt <121>, USP 23 / NF 18 1995) . Of course, other suitable assays of the hormonal activity of insulin are known in the art, and may be used if desired to determine the level of hormonal effectiveness or ineffectivenesε of a particular preparation of one or more inεulin related polypeptideε.
Hormonally ineffective inεulin polypeptideε εuitable for use in the practice of the present invention can be prepared by conventional meanε well known in the art, including: purification from natural εourceε (i.e., from animal pancreases, e.g. porcine pancreases) and subεequent denaturation (and chain εeparation if deεired) ; preparation by chemical synthetic means εuch aε solid phase synthesis or the like, and preparation by conventional recombinant DNA techniques (e.g., microbial, insect or mammalian cell synthesiε directed by a plasmid vector comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence corresponding to at least part of the amino acid sequence of a hormonally effective inεulin) followed by conventional purification of the hormonally ineffective inεulin. Alternatively, hormonally effective insulinε may be prepared by any of the various means known in the art, and may then be inactivated, preferably by denaturation (followed by chain separation, if desired) .
Denaturation of hormonally effective insulinε to produce hormonally ineffective insulin polypeptides may be carried out by any method of denaturation, a large variety of which are known to those of skill in the art. These include heat denaturation, various types of chemical denaturation (e.g., using chaotropic agents such as urea or guanidinium HCl, or other chemical means of denaturation such as carboxymethylation) . Denaturation of inεulin related polypeptideε produced by εynthetic or recombinant DNA methodε may alεo be achieved by εequence truncation or inεulin precursor εynthesiε in organiεmε incapable of proceεεing the precursor to produce mature, hormonally effective insulin.
As discussed above, combinations of these hormonally ineffective insulin related polypeptides may also be uεed in the practice of the preεent invention as long as their combination does not generate a hormonally effective inεulin polypeptide. Aε used herein, a hormonally ineffective insulin related polypeptide has leεε hormonal activity than the minimum activity for insulin required by the United States Pharmacopea (USP 23 / NF 18, 1995) , which is 26 USP units per milligram. Therefore, in accordance with the present invention, any insulin related polypeptide exhibiting 25 USP unitε of inεulin activity per milligram or leεε iε considered to by hormonally ineffective. Preferably the hormonally ineffective insulin related polypeptide has lesε than 20 USP unitε of activity per mg, more preferably less than 10 USP units per mg, and most preferably lesε than 5 USP unitε of inεulin activity per mg of polypeptide. Superphysiologic Doses of Hormonally Ineffective Insulin Polypep ides
As discussed above, doses containing superphysiologic amounts of hormonally ineffective insulin related polypeptideε meanε doses containing amounts providing at least an equivalent number of moleε of hormonally ineffective insulin related polypeptide to the number of moles of hormonally effective insulin that will induce insulin εhock in the patient. In accordance with the present invention, such superphyεiologic amounts of hormonally ineffective insulin related polypeptideε compriεe amounts ranging from about 6.9 pM/kg/patient to about 8.6 μM/kg/patient. Preferably the amounts range from about 34.5 pM/kg/patient to about 5.2 μM/kg/patient. More preferably the amounts range from about 170 pM/kg/patient to about 3.5 μM/kg/patient. Most preferably the amounts range from about 0.5 μM/kg/patient to about 3.5 μM/kg/patient.
In certain of its aspectε, the present invention also provides for the repeated adjuvant free administration of doses containing lower (not superphyεiologic) amounts of hormonally ineffective insulin related polypeptides to a patient in need of εuch treatment. Although less preferred, doses containing amounts of below about 6.9 pM/kg/patient, and as low as about 1 pM/kg/patient may be uεed in the practice of the preεent invention. Aε referred to herein and in the claimε, εuch lower amounts are referred to as "physiologic doses" or "physiologic amounts" when they contain a number of moles of hormonally ineffective insulin related polypeptide that is less than the number of moles of hormonally effective insulin that will induce insulin shock in the patient, but contain at least about 1 picomole of hormonally ineffective insulin related polypeptide, i.e., at least as many moleε of inεulin related polypeptide as there are moles of insulin in about 0.15 USP units of insulin with a specific activity equal to the USP minimum activity standard of 26 units per mg (the equivalent of about 5.8 ug of human insulin with that specific activity) . Administration on Therapeutic T Cell Modulatory Schedules
In accordance with the present invention, a therapeutic T cell modulatory schedule involves administration of doses containing superphyεiologic amounts of the hormonally ineffective inεulin polypeptideε repeatedly to the patient at least two times at an interval of at least six, and preferably at least twelve hours and not more than seven days between doses. In accordance with the present invention, the polypeptides are administered parenterally without the concomitant administration of an adjuvant. Administration by a parenteral route will typically be via injection such as intravascular injection (e.g., intravenous infusion), subcutaneous injection, or intramuscular injection. Other non- oral routes of administration, e.g., mucosal, inhalation, transdermal ultrasound, and the like, may be used if desired and practicable for the particular hormonally ineffective insulin related polypeptide to be administered.
Formulations suitable for injection and other routes of administration are well known in the art and may be found, for example, in Remington's Pharmaceutical Sciences. Mack Publiεhing Company, Philadelphia, PA, 17th ed. (1985) . Preferred formulations for parenteral administration of hormonally ineffective insulin related polypeptides are those described for insulin in the USP 23/NF 18 (1995) .
Parenteral formulations muεt be εterile and non-pyrogenic, and generally will include a pharmaceutically effective carrier, εuch aε saline, buffered (e.g., phosphate buffered) saline, Hank'ε εolution, Ringer's solution, dextrose/saline, glucose solutions, and the like. Formulations may alεo contain pharmaceutically acceptable auxiliary εubεtances as required, such as, tonicity adjusting agentε, wetting agentε, bactericidal agents, preservatives, stabilizers, and the like.
Without intending to limit it in any manner, the present invention will be more fully described by the following examples.
Experimental Methods
Non-obese diabetic (NOD) mice are a strain of mice that are prone to the development of diabetes, and repreεent an accepted model system for the study of diabetes mellitus. Theεe mice, deεignated NOD/MrkTacfBR, were purchaεed from Taconic Farmε
(Germantown, NY) . NOD SCID mice, deεignated NOD/SCID, are available from the Jackεon Laboratorieε, Bar Harbor, ME. The incidence of diabeteε by 200 dayε of age in these mice is 80% in females and 50% in males. At 110 days of age, fewer than 15% of the male NOD mice became diabetic.
CYTOXAN induction experiments: CYTOXAN (Cyclophosphamide) treatment can increase the incidence and accelerate the onset of diabetes in non-diabetic NOD mice. Randomly selected, non- diabetic male NOD mice, which were 100 to 110 days of age, were injected (ip) with cyclophosphamide (250mg/kg) on day 0.
All the mice were subjected to urine glucoεe meaεurement 2- 3 times per week for a period of 21 days after cyclophosphamide injection. The onset of diabetes was recorded as the first point at which two consecutive days of poεitive urine glucose resultε were obtained. Blood glucose was also measured to confirm the urine glucose reεults. (ExacTech, MediSense Inc., Cambridge, MA) In all the mice tested with poεitive urine glucoεe, blood glucoεe levelε were greater than 150mg/dl. At day 21, theεe mice were εacrificed and examined hiεtologically.
Adoptive transfer experiments: Spleen mononuclear cells were harvested from newly onεet diabetic NOD mice (diabetic not more than 3 weekε) and 35 x 10^ spleen cells in 0.2 ml of PBS were injected intravenouεly into 6-8 weekε old NOD SCID mice. Onset of diabetes were monitored biweekly by urine glucose teεting and confirmed by blood glucoεe testing on day 28 relative to the time of spleen cell transfer. At day 28, these mice were sacrificed and examined histologically. Reagents;
CYTOXAN (cyclophosphamide, Mead Johnson oncology products) waε diεεolved in distilled water (25mg/ml) . Oxidized bovine insulin Chain B was purchased from Sigma (catalog no. 1-6383) . Both insulin chain B and the BSA control protein (Miles Inc., #81-001) were dissolved in PBS/1M HCL, pH2, before dialysiε against PBS, pH7.2, sterile filtration, and storage of frozen aliquots. Treatment;
Inεulin Chain B treatment in the CYTOXAN model: Groups of randomly selected NOD mice were injected intravenously twice daily with either 500ug insulin Chain B or 500ug BSA on dayε 1, 3, and 5 following CYTOXAN treatment (day 0) . Inεulin Chain B treatment in the adoptive tranεfer model: NOD/SCID mice were injected intravenously twice daily with 500ug of inεulin Chain B (or BSA as control) on day 3, 5 and 7 relative to the time of spleen cell transfer (day 0) .
Although preferred and other embodiments of the invention have been described herein, further embodiments may be perceived by those skilled in the art without departing from the scope of the invention as defined by the following claims.
Table-1: doεe dependency of cyclophosphamide induced diabetes in non-diabetic male NOD recipients
CYTOXAN # Diabetic / Total % Mean onset time
200mg/kg 2 / 7 28.6% 12
250mg/kg 5 / 7 71.4% 11
300mg/kg 5 / 7 71.4% 12.6

Claims

What is claimed is:
1. A method of treating a patient selected from the group consisting of patients at riεk of developing type 1 diabetes and patients suffering from type 1 diabetes, said method comprising the repeated administration of a dose of a measured amount of a hormonally ineffective insulin related polypeptide to said patient.
2. The method of Claim 1 wherein the amount is a superphysiologic amount.
3. The method of Claim 1 wherein the amount of the hormonally ineffective insulin related polypeptide is an amount that is effective, after at least two doseε, to reduce the patient ' ε serum levels of at leaεt one type of circulating autoantibody, εaid type of autoantibody selected from the group consisting of ICA, IAA, and GAD autoantibodies.
4. The method of Claim 1 wherein the amount is a physiologic amount.
5. The method of Claim 1 wherein the amount is greater than or equal to about 6.9 pM per kilogram per patient per dose and less than or equal to about 8.6 μM per kilogram per patient per dose.
6. The method of Claim 5 wherein the amount is greater than or equal to 0.5 μN per kilogram per patient per dose and less than or equal to 3.5 μM per kilogram per patient per dose.
7. The method of Claim 1 wherein the repeated administration comprises administration of at least two doses at an interval of not less than twelve hours and not more than 24 hours.
8. The method of Claim 1 wherein the insulin related polypeptide is insulin Chain B.
9. The method of Claim 1 wherein the insulin related polypeptide is insulin Chain A.
PCT/US1996/014348 1995-09-06 1996-09-05 Treatment of diabetes via administration of hormonally ineffective insulin WO1997009061A1 (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
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WO1992006704A1 (en) * 1990-10-10 1992-04-30 Autoimmune, Inc. Method of treating or preventing type 1 diabetes by oral administration of insulin
US5422339A (en) * 1991-03-19 1995-06-06 Joslin Diabetes Center, Inc. Peptides having insulin autoantibody but not insulin receptor binding capacity

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Title
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CLINICAL RESEARCH, 1983, Vol. 31, No. 2, SHIPP et al., "Severe Insulin Resistance in Patient With Type I Diabetes Responsive Only to Intraperitoneal Insulin: A New Syndrome?", page 546A. *
DIABETES, May 1985, Volume 34, Supplement 1, NIPPE et al., "Intraperitoneal (IP) Versus Intravenous (IV) Application of Human Proinsulin and Insulin in Type I Diabetics", page 59A, Abstract 236. *
JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, 1984, Vol. 58, No. 6, BERGENSTAL et al., "The Metabolic Effects of Biosynthetic Human Proinsulin in Individuals With Type I Diabetes", pages 973-979. *

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