1. FIELD OF THE INVENTION
The present invention relates to a process for ameliorating or preventing neurological diseases that are caused, in part, by an increased and/or abnormal responsivity to interferon. Down Syndrome (DS) and Alzheimer's Disease (AD) are examples of such diseases. Specifically, the invention provides a method for treating subjects suffering from or at risk for such diseases by the administration of a pharmacological preparation that antagonizes interferons' action.
2. BACKGROUND OF THE INVENTION
2.1. The Molecular Biology of Interferons and Interferon Receptors
Interferons are proteins that alter and regulate the transcription of genes within a cell by binding to interferon receptors on the regulated cell's surface and thus prevent viral replication within the cells. There are five types of interferons (IFN), which are designated α (formerly α1), ω (formerly α2), β, γ and τ. Mature human interferons are between 165 and 172 amino acids in length. In humans IFN-α and IFN-ω are encoded by multiple, closely related non-allelic genes. Additionally, there are pseudo-genes of IFN-α and IFN-ω. By contrast, IFN-β and IFN-γ are encoded by unique genes.
The interferons can be grouped into two types. IFN-γ is the sole type II interferon; all others are type I interferons. Type I and type II interferons differ in gene structure (type II interferon genes have three exons, type I one), chromosome location (in humans, type II is located on chromosome-12; the type I interferon genes are linked and on chromosome-9), and the types of tissues where they are produced (type I interferons are synthesized ubiquitously, type II by lymphocytes). Type I interferons competitively inhibit each others binding to cellular receptors, while type II interferon has a distinct receptor. Reviewed by Sen, G. C. & Lengyel, P., 1992, J. Biol. Chem. 267:5017-5020.
Although all type I interferons compete for binding to a common receptor or receptors, the effects of different type I interferons can be different. Pontzer, C. H., 1994, J. Interfer. Res. 14:133-41. Additionally, there appears to be several kinds of type I interferon receptor. For example, there is evidence that the type I interferon receptors of different cell types are different. Benoit, P., 1993, J. Immunol. 150:707. The number of genes encoding the type I interferon receptors is unknown: however, the genes appear to be linked to each other and to at least one gene encoding an IFN-γ receptor component as well. In humans, chromosome region 21q21.1-21.31 encodes all the genes needed for the receptor for type I interferon (Raziuddin, A., 1984, Proc. Natl. Acad. Sci. 81:5504-08; Soh, J., 1993, Proc. Natl. Acad. Sci. 90:8737-41; Soh, J., 1994, J. Biol. Chem. 269:18102-10) and at least one essential component of the type II interferon receptor (Jung, V., 1990, J. Biol. Chem. 265:1827-30).
2.2. The Biology of Interferon Action and Down Syndrome
The binding of interferons to their receptor, leads to a cascade post-translational modification to other proteins which are then transported to the nucleus where they regulate the transcription of genes by binding to specific nucleic acid sequences. The nucleic acid sequence which is characteristic of genes responsive to type I interferons is designated the Interferon Sensitive Response Element (ISRE). Reviewed Tanaka, T. & Taniguchi, T., 1992, Adv. Immunol. 52:263. Type-I interferons are synthesized in response to viral infection, except for IFN-τ which is constitutively produced in the placenta; Type II interferons are synthesized in response to antigen stimulation.
Interferons alter the rates of synthesis and the steady state levels of many cellular proteins. An overall effect of interferon is usually an inhibition of cellular proliferation.
The possibility that cells from subjects having Down Syndrome may have abnormal responsivity to interferon was introduced by the discovery that a gene encoding an interferon inducible protein, which was subsequently identified as the type I interferon receptor, was located on chromosome-21. Tan Y. H. et al., 1974, J. Exp. Med. 137:317-330. This observation prompted comparisons of the response of diploid and trisomy-21 aneuploid cultured cells to interferon added to the culture medium. These studies have consistently shown an increased responsivity of trisomy-21 cells to interferon. Tan, Y. H., et al., 1974, Science 186:61-63; Maroun, L. E., 1979, J. Biochem. 179:221; Weil, J., et al., 1983, Hum. Genetics 65:108-111; reviewed Epstein, C. J., & Epstein, L. B., 8 LYMPHOKINES pp277-301 (Academic Press, NY, 1983); Epstein, C. J. et al., 1987, ONCOLOGY AND IMMUNOLOGY OF DOWN SYNDROME (Alan R. Liss, 1987). The publications of these studies have been accompanied by speculative conjectures that the altered responsivity to interferon played a role in the pathogenesis of lesions of Down Syndrome. See, Maroun, L. E., 1980, J. Theoret. Biol. 86:603-606.
2.3. Down Syndrome and Animal Models of It
An animal model of Down Syndrome has been constructed by use of the knowledge that human chromosome-21 is syntenic to mouse chromosome-16, i.e., that many of the genes present on each are homologs of each other. Mice having specified trisomies can be bred by use of parental mice having “Robertsonian” chromosomes, i.e., chromosomes that are essentially the centromeric fusion of two different murine chromosomes. A variety of such Robertsonian chromosomes have been identified, including at least two involving chromosome-16 and a second different chromosome: Rb(16.17) and Rb(6.16). Mice homozygous for any Robertsonian or combination of independent Robertsonian chromosomes are euploid and fertile. The intercross (F1) between an Rb(16.17) and an Rb(6.16) mouse is also fully diploid at each genetic locus, although errors in meiosis may cause reduced fertility. Note that in such an F1 both the maternal and paternal chromosome-16 are a part of a Robertsonian chromosome.
Because of meiotic errors the outcross between a mouse having both two different Robertsonian chromosome-16's and a non-Robertsonian mouse gives rise to a trisomy-16 conceptus in between 15% and 20% of cases. Gearhart, J. D., et al., 1986, Brain Res. Bull. 16:789-801; Gropp, A., et al., 1975, Cytogenet. Cell Genet. 14:42-62. The murine trisomy-16 fetuses develop to term but do not live beyond birth by more than a few hours.
Examination of the fetal trisomy-16 and the post-partum human trisomy-21 reveals a number of analogous or parallel lesions. For this reason, the murine trisomy-16 construct-is considered to be an animal model of Down Syndrome. Epstein, C. J., THE METABOLIC BASIS OF INHERITED DISEASE, 6TH ED. pp291-326 (McGraw-Hill, NY, 1989); Epstein, C. J., et al., 1985, Ann, N. Y. Acad. Sci. 450:157-168. Because a murine trisomy-16 fetus is not viable post partum, the opportunity to study the neurological pathology of the model has been limited. However, it is clear that in both human trisomy-21 and murine trisomy-16 there is an overall reduction in fetal size and particularly in the development of the fetal brain. Epstein, C. J., THE CONSEQUENCES OF CHROMOSOME IMBALANCE: PRINCIPLES, MECHANISMS AND MODELS (Cambridge University Press, NY, 1986). Further insights into the effects of murine trisomy-16 have been obtained by the formation of Ts16++2N chimeras (Gearhart, J. D., et al., 1986, Brain Res. Bulletin 16:815-24) and by transplantation of fetal-derived Ts16 tissue into a 2N host (Holtzman, D. M., et al., 1992, Proc. Natl. Acad. Sci. 89:1383-87; Holtzman, D. M., et al., DOWN SYNDROME AND ALZHEIMER DISEASE, pp227-44 (Wiley-Liss, NY, 1992).
2.4. Alzheimer'S Disease and Amyloid Precursor Protein
Alzheimer's Disease is a progressive dementia which is characterized by the precipitation of a peptide, termed an Aβ peptide, of about 40 amino acids within the brain and within the walls of blood vessels in the brain. The Aβ peptide is derived from the processing of a larger cell surface protein called the β Amyloid Precursor Protein (βAPP). Production of the Aβ peptide is not per se pathological. The functions of both the Aβ peptide or βAPP are unknown.
Several lines of evidence indicate that the deposition of the Aβ peptide is not merely correlative but rather causative of Alzheimer's Disease. The gene encoding βAPP is located on chromosome-21 and, as noted above, subjects having Down Syndrome develop Alzheimer's Disease. More directly, kinship groups have been identified among the many causes of familial Alzheimer's Disease in which the inheritance of the Disease is linked to the inheritance of a gene encoding a mutated βAPP, moreover the mutation is within the Aβ peptide itself. Reviewed Selkoe, D. J., 1994, Ann. Rev. Neurosci. 17:489-517. Transgenic mice, having multiple copies of such a mutant βAPP gene, operatively linked to a strong, neuronal and glial cell specific promoter, develop the anatomical lesions of Alzheimer's Disease at about 6-9 months of age. Games, D., et al., 1995, Nature 373:523.
There is a relationship between Down Syndrome and Alzheimer's Disease. The gene encoding the βAPP is found on chromosome-21. Patients with Down Syndrome are at increased risk of developing Alzheimer's Disease, most often by about the fifth decade of life although cases of earlier development have been reported. Mann, D. M. A., et al., 1990, Acta Neuropathol. 80:318-27.
3. SUMMARY OF THE INVENTION
The present invention is based, in part, on the recognition that in certain pathologic processes that result in mental impairment, the host is rendered abnormally and/or aberrantly sensitive to the effects of interferon so that the effects of interferon become an immediate and direct cause of the pathology. Such processes include, in humans, trisomy of chromosome-21 or the portion of the chromosome-21 that encodes the receptor for type I interferon and at least one component of the receptor for IFN-γ, which is the genetic abnormality associated with Down Syndrome; and also include Alzheimer's Disease.
The present invention provides a method of ameliorating the pathologic effects of interferon by administering to a subject, in the above-noted circumstances, an antagonist of interferon. Embodiments of the invention include the administration of antagonists, alone or in combination, that are antagonists of Type I interferon, Type II interferon (IFN-γ), and placental interferon (IFN-τ).
4. DESCRIPTION OF THE FIGURES
FIG. 1A-C. The lengths of Trisomy 16 fetuses plotted as a function of the average length of normal littermates.
FIG. 1A, Uninjected controls; FIG. 1B, non-specific IgG (ns-IgG) injected controls; FIG. 1C, anti-IFN injected fetuses. An analysis-of-covariance was performed to compare the groups on length while adjusting for average normal littermate length. The lengths of the anti-IFN treated group were significantly greater than those of the ns-IgG injected controls (p=0.0112) and those of the uninjected controls (p=0.0037). The dotted lines in each figure encompass the 95% confidence limits.
FIG. 2A-B. Morphometric analysis of the development in normal, Trisomy 16 treated and Trisomy 16 sham treated fetuses. FIG. 2A, average eye opening of 17 to 23 mm trisomy 16 fetuses; FIG. 2B, average back curvature scores of trisomy 16 fetuses greater than 20 mm in length. Columns: (A) Uninjected; (B) non-specific IgG injected; (C) anti-IFN injected; (D) euploid. The mean ± standard error is presented.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Selection of Subjects
The present invention concerns the administration of interferon antagonists to subjects in order to ameliorate the neurological and developmental abnormalities in the subject due to the action of interferon. A particular group of subjects at risk are subjects having a trisomy of the portion of the chromosome region, designated in humans 21q21.1-21.31, that encodes for interferon receptors. This group has the clinical diagnosis of Down Syndrome. Grete, N., 1993, Eur. J. Hum. Genetics 1:51-63; Sinet, P. M., 1994, Biomed. & Pharmacol. 48:247-252. The homologous chromosome in mice is chromosome-16.
Diagnosis of Down Syndrome can be made by any method known to the medical arts. Typically, for diagnosis in utero, amniocentesis can be performed at about 14 weeks of gestational age and chorionic villus sampling (biopsy) can be performed between 9 and 12 weeks of gestational age. Down Syndrome in children and adults is diagnosed from karyotypes of peripheral blood cells. Cells from either type of sample are cultured and cytogenetic examination can be performed by methods well understood by those skilled in the art.
As noted above, subjects having Down Syndrome are at increased risk to develop Alzheimer's Disease. A further group of subjects that would benefit from the invention consist of subjects having the diagnosis of probable Alzheimer's Disease or who are at increased risk of developing Alzheimer's Disease from causes other than Down Syndrome. The diagnosis of probable Alzheimer's Disease is made by clinical criteria (McKhann, G., 1984, Neurology 34:939; DIAGNOSTIC AND STATISTICAL MANUAL OF MENTAL DISORDERS IV, American Psychological Association, Washington, D.C.). Persons having a familial predisposition to Alzheimer's Disease are also suitable subjects for the present invention.
5.2. The Selection of Antagonists
The antagonist of the invention can be any antagonist that can be administered to the subject in an amount effective to prevent the deleterious action of the interferon on the central nervous system.
The effective amount of antagonists that act by binding to and blocking interferon proteins in the blood can be determined by assaying the concentration of bioavailable interferon in the subjects blood. An effective dose of antagonist is a dose that is sufficient to reduce the level of bioavailable interferon by between at least three to five fold, more preferably by about ten fold and most preferably by about twenty five fold below the normal levels of interferon.
The assay of bioavailable interferon is performed by adding a sample of the subjects blood to a culture of an interferon sensitive cell line which is then infected with a test virus, typically Vesicular Stomatitis Virus (VSV), and the number of viral plaques is determined or the cytotoxic effects of the VSV infection is otherwise quantitated. Bioavailable interferon blocks productive viral infection. The level of bioavailable interferon is calculated by comparing various dilutions of the test sample with a titration of a standard sample of interferon. Such assays are rountine in the art. See, e.g., Hahn, T., et al., 1980, in INTERFERON: PROPERTIES AND CLINICAL USES, ed. by A. Khan, N. O. Hill and G. L. Dorn, (Leland Fikes Foundation Press, Dallas, Tex.); Armstrong, J. A., 1971, Applied Microbiology 21:723-725; Havell, E. A. & Vilcek, J. 1972, Anti-microbial Agents and Chemotherapy 2:476-484.
In one embodiment of the invention the antagonist is a monoclonal anti-interferon antibody or fragment thereof. The production of such antibodies is well known in the art. The production of anti-IFN-α monoclonal antibodies that block interferon activity is taught by U.S. Pat. No. 4,973,556 to Bove et al. The production of blocking monoclonal antibodies to IFN-γ is taught by U.S. Pat. No. 4,948,738 to Banchereau. The structure of human trophoblastic interferon (IFN-τ) has been recently disclosed (Whaley, A. E., 1994, J. Biol. Chem. 269:10864-8). Monoclonal antibodies and other antagonists to this interferon can be produced using methods well known to those skilled in the art.
In a preferred embodiment, the antibody is a “chimeric” antibody, i.e., an antibody having a variable region from one species and a constant region from another species. Most typically chimeric antibodies for use in humans have constant regions of human origin. In an alternative preferred embodiment, the antibody is a “grafted” antibody, i.e., an antibody having complementarity determining regions from one species and a constant region and a framework region of the variable region from a second species. A grafted antibody in which the second species is human is termed a “humanized” antibody. Methods of making chimeric antibodies suitable for pharmaceutical use are disclosed in patent publication W092/16553 by Le, J. (Oct. 1, 1992). “Grafted” antibodies and “humanized” antibodies are described in U.S. Pat. No. 5,225,539 to Winter and patent publications W091/09967 and W092/11383 by Adair, J. R. et al. Suitable antagonists, smaller than an antibody molecule, can be derived from anti-interferon monoclonal antibodies by techniques well known in the art. See, e.g., U.S. Pat. No. 5,091,513 to Huston and U.S. Pat. No. 5,260,203 to Ladner. As used herein the term “antibody antagonists” includes natural polyclonal and monoclonal antibodies, chimeric and grafted antibodies, and enzymatically and recombinantly produced interferon binding fragments of each type of antibody.
In an alternative embodiment the antagonist can be a recombinantly produced protein that comprises the interferon binding portion of an interferon receptor. The production of soluble interferon receptors by baculovirus transduced cells is described in Fountoulakis et al., 1991, Eur. J. Biochem. 198:441-450. Alternatively the antagonist can be a fusion protein that contains an interferon binding domain of an interferon receptor.
In alternative embodiments, the antagonist can be an antibody to an interferon receptor, a soluble interferon receptor, receptor fragment, or a peptide that is derived from an interferon that occupies the receptor binding site but does not activate the receptor. Such an IFN-γ peptide antagonist is disclosed by Jarpe, M. A. et al., 1993, J. Interferon Res. 13:99-103.
When the subject is a fetus, or an infant less than 6 weeks of age, the blood brain barrier is not fully formed. In these circumstances antibodies and other proteins that block the interferon receptor can directly reach the central nervous system. When the subject has an intact blood brain barrier, the preferred embodiment of the invention employs antibodies and proteins that block interferon by binding the interferon directly, rather than those that act at the interferon receptor.
Alternatively, increased CNS entry of antibody antagonists can be obtained by chemical modification of the antagonist. Such modifications include cationization, Pardridge, W., 1991, “Peptide Drug Delivery to the Brain”, and glycation, Poduslo, J. F., & Curran, G. L., 1994, Molecular Brain Research 23:157.
The interferon antagonist can be a mixture of antagonists that are specific for the various different types of interferon. When one type of interferon predominates, the antagonist can be an antagonist for only the predominate type of interferon that is present. For example, when the subject is a fetus, the antagonist can be a INF-τ specific antagonist.
When the subject is a fetus, then the antagonist can be administered by a transplacental route, e.g., antibody that is transported across the placenta. The human isotypes IgG1, IgG3 and IgG4 are suitable for transplacental administration.
5.3. Selection of Dose and Timing of Administration
The amount of an antibody antagonist administered is between 1 and 100 mg/kg. The preferred route of administration of an antibody antagonist is intravenous administration to infant and adult subjects. The preferred route of administration to fetal subjects is by intravenous administration to the mother followed by transplacental transport. Alternatively antibody antagonists can be administered by intramuscular and subcutaneous routes. When an antagonist is delivered transplacentally, the calculation of the dose is based on the maternal weight.
The antagonist is administered to subjects having Down Syndrome preferably at the time when the central nervous system is developing most rapidly. The preferred period of administration is from a gestational age of 24 weeks onwards until a post natal age of about 2 years. Even though some proliferation of neurons takes place during weeks 8-18, it is not critical that an antagonist be administered to a human subject prior to week 20-24 of gestational age because the synaptic connections between the neurons are not formed until week 20. Brandt, I., 1981, J. Perinat. Med. 9:3. The administration of the antagonist to subjects having Alzheimer's Disease should commence at the time that the diagnosis of probable Alzheimer's Disease is first made and continue there after. In middle age, subjects having Down Syndrome develop a dementia having an anatomical pathology which is identical to Alzheimer's Disease (Mann, D. M. A., 1988, Mech. Aging and Develop. 43:99-136). Thus, the administration of the antagonist to Down Syndrome patients can be continued throughout the life of the patient, as Down Syndrome patients are at risk for Alzheimer's Disease ab initia.
The frequency of administration is determined by the circulation time of the antagonist, which can be determined by direct measurement by methods well known to those skilled in the art.
In an alternative embodiment of the invention, the administration of interferon antagonists is replaced by the extracorporeal treatments of the subject's blood to remove circulating interferon, such as is described in U.S. Pat. No. 4,605,394.
5.4. A Model Embodiment of the Invention
The invention is exemplified and its operability is demonstrated by the experiments that are presented in Example 1 below. Briefly, normal female mice were crossed with double heterozygous males having Rb(6.16) and Rb(16.17) chromosomes. The females were injected with a mixture of rat monoclonal anti-IFN-γ (1500 neutralizing units) and rabbit polyclonal anti-IFN-α/β (1362 neutralizing units) interperitonally (i.p.) on days 8, 10, 12 and 14 of pregnancy. On day 17 the embryos were biopsied for cytogenetic classification, sacrificed and four gross parameters were measured and compared to the genetically normal littermates in order to assess relative development. Control groups consisted of untreated females and sham treated females which were given normal rabbit and rat serum γglobulin injections.
The four measured parameters were overall (crown-rump) length of the fetus, shape of the back (normally concave at birth), eye-closing (the eyes normally close shortly before birth) and fetal weight. The results of the comparison of each of the parameters from 17 untreated, 16 sham treated and 18 treated controls showed a statistically significant reduction in the growth retardation/maturation of the treated trisomy-16 fetal mice compared to their euploid littermates.
The fetuses from anti-IFN treated mothers had a mean weight decrease of −10.92% compared to a −21.47% decrease for the uninjected group (p=0.079) and a −30.46% decrease for the ns-IgG injected group (p=0.0003) relative to diploid littermates. The uninjected and ns-IgG injected control groups were not statistically different from each other (p=0.174).