WO1989007449A1 - Methods for treating immunoinflammatory and other disease conditions - Google Patents

Abstract

Pharmaceutical compositions comprising compounds related to the peptide Lys-Ser, and therapeutic methods of utilizing such compositions in the prevention or treatment of mammalian immunoinflammatory diseases and other disease conditions are described.

Description

METHODS FOR TREATING IMMUNOINFLAMMATORY AND OTHER DISEASE CONDITIONS

Related Applications

This application corresponds and claims priority to U.S. Patent Application Serial No. 157,183, filed February 16, 1988. That application is a continuation-in- part of U.S. Serial No. 803,452, filed November 29, 1985, which is a continuation-in-part of U.S. Serial No. 692,711 (filed January 18, 1985 and now abandoned), and is also a continuation-in-part of U.S. Serial No. 764,294 (filed August 9, 1985 and now abandoned). Serial No. 692,711 is > in turn a continuation-in-part of U.S. Serial No. 522,602 (filed August 12, 1983, now U.S. Patent No. 4,683,292). Serial No. 764,294 is a continuation-in-part of U.S. Serial No. 746,175 (filed June 18, 1985 and now aban¬ doned), which is in turn a continuation-in-part of U.S. Serial No. 522,601 (filed August 12, 1983 and now aban¬ doned) ; Serial No. 764,294 is also a continuation-in-part of U.S Serial Nos. 522,602, 522,738 (now Patent No. 4,686,282) and 522,739 (now Patent No. 4,579,840) (all filed August 12, 1983). Serial No. 764,294 also corresponds to Republic of South Africa Patent Application Serial No. 84/6192 (filed August 9, 1984). The entire specification, claims, drawings and abstract of each of the patents and patent applications named above are incorporated herein by reference.

Background Of The Invention

A large number of diseases are believed to occur because of a regulatory imbalance in the immune system. Autoimmune diseases are an example of conditions in which a substantial portion of an immune response is directed toward healthy host cells. Under normal conditions, the immune system exhibits tolerance toward cells of the host which prevents the immune system from attacking normal. healthy cells. It is this critical ability of the immune system to distinguish "self" from foreign cells and molecules that provides selectivity of an immune system attack. In autoimmune diseases, tolerance for host cells ^" and molecules is reduced or eliminated resulting in significant destruction of otherwise healthy cells and organs.

The tendency of a host's immune system to display reduced tolerance to normal cells is strongly influenced by cell surface molecules whose genes are associated with the host's major histocompatibility complex (MHC) . A particular MHC haplo-type may substantially increase the risk of sel -tolerance loss and subsequent autoimmunity. In certain autoimmune diseases, infection by certain viruses or bacteria is believed to trigger the loss of self-tolerance which, in the setting of an appropriate MHC haplotype, results in an autoimmune disease. In other autoimmune diseases the triggering events which lead to a loss of sel -tolerance remain unknown. Autoimmune diseases may affect every organ of the body. Examples of diseases thought to have an autoimmune pathogenesis include, but are not limited to, autoimmune arthritis including rheumatoid arthritis and psoriatic arthritis, autoimmune neurologic disease including Ξ multiple sclerosis and postviral encephalomyelitis, systemic lupus erythematosus, ankylosing spondylitis, Reiter's syndrome, Sjogren's syndrome, polymyositis- dermatomyositis, thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, regional enteritis (Crohn's disease), chronic active hepatitis, primary biliary cirrhosis, idiopathic interstitial pulmonary fibrosis, Goodpasture*s syndrome, Guillain-Barre syndrome, myasthenia gravis. Grave's disease, Hashimoto's thyroid- itis, juvenile onset insulin-dependent diabetes, Addison's ^" disease, pernicious anemia, pemphigus, bullous pemphigoid and other diseases and conditions. The immune response responsible for the rejection of transplanted organs among genetically non-identical animals or humans in many ways resembles an autoimmune disease in that an otherwise healthy transplanted organ may be destroyed by the recipient's immune system. Such destruction occurs because the recipient's immune system recognizes the "foreign" histocompatibility antigens present on cells of the transplanted organ and trigger a destructive immune response. Similarly, the transplant into a recipient of graft material may lead to a graft- versus-host response and graft-versus-host disease (GVHD) wherein transplanted T lymphocytes initiate a destructive immunologic reaction against the host's cells, particular¬ ly host spleen cells. The immune system disorders noted above are generally characterized by the existence of a deleterious immuno- inflammatory condition, i.e., an inflammatory condition caused or mediated by an inappropriate immune response. Such conditions will be referred to herein as mammalian im unoinflammatory diseases.

The complex regulation of immune responsiveness results from interactions between all classes of leuko¬ cytes, molecules secreted by leukocytes and cells and molecules from other organ systems. One class of leuko- cytes in particular, termed thymus-derived lymphocytes or T lymphocytes (T cells) , is considered to be critically important to the coordination and regulation of most immune responses. T cells may be divided into various sub-sets which have distinct immune functions. Helper T cells, for example, are critical for the growth and development of B cells into antibody-secreting plasma cells. Helper T cells are also critical for the growth and development of other T cell subsets such as Killer T cells which can directly destroy infectious agents, cancer cells, transplanted organs and, in autoimmune disease, healthy cells. By contrast, other T cell subsets termed suppressor T cells actively suppress the growth and development of B cells. Killer T cells and other lymphoid cells. Suppressor T cells are also critical for the development and maintenance of immune tolerance that prevents the development of autoimmune disease and organ 5 transplantation rejection.

Because T cells have a powerful influence on the induction or suppression of the immune responses that lead to autoimmunity and organ transplantation rejection, pharmacologic agents which regulate T cell functions may

Itt provide significant therapeutic benefit in the treatment of human or animal disease. As discussed in more detail below, the present invention is directed to the treatment of immunoinflammation thought to be mediated by T cells in diseases including multiple sclerosis, rheumatoid

15 arthritis, organ transplantation rejection, graft-versus- host disease and other diseases. The ability of the described compounds to suppress T cell activity is thought to be one aspect of the mechanism leading to their utility.

20 There are certain diseases in which unregulated overproduction of antibodies produce a major portion of the clinical and pathological abnormalities present. Systemic lupus erythematosus (SLE) is the most common of such conditions and afflicts over 500,000 patients in the

ZS United States. While the cause of SLE is not known, the precise mechanism by which the disease produces inflammation and organ destruction is understood.

Patients with SLE have an abnormality in the mechanisms that regulate antibody synthesis. Healthy

30 humans have the capacity to synthesize antibodies to virtually any infectious or "foreign" material to which they are exposed. Such synthesis does not normally occur, however, until the person is appropriately infected or otherwise exposed to the agent. SLE patients, by

35 contrast, have their antibody synthesizing machinery operating at a high capacity and synthesize antibodies directed towards a broad range of entities, their own cells and organs included. Not surprisingly, this antibody "autoimmune" response produces considerable inflammation and frequently results in the destruction of entire organs, especially kidneys, which become inflamed during the process of filtering antibody complexes from the blood.

B lymphocytes (B cells) are responsible for the synthesis of all antibodies. Normal antibody synthesis is largely under the regulatory control of T cells which direct the appropriate immature B cell to develop into an antibody-secreting cell. In one approach to treating antibody-mediated diseases such as SLE, regulatory T cells could be stimulated by a peptide to regain regulatory control of the unregulated B cells. A second approach includes the use of a peptide to regulate and suppress B cell function directly, thus suppressing antibody synthesis.

Peptides capable of regulating B cell activity would have therapeutic applications in diseases other than SLE as well. Rheumatoid arthritis patients also suffer from over-synthesis of antibodies which produce inflammation and cell destruction by mechanisms similar to that in SLE.

Myesthenia gravis represents a third disease in which virtually all symptoms are directly due to abnormal over- synthesis of a particular antibody directed towards a neurotransmitter receptor of muscles. Certain types of autoimmune anemia result also result from abnormal synthesis directed toward healthy red blood cells. These diseases, and others, represent a large category of patients for whom conventional therapies are poor and are frequently toxic in their own right.

While many diseases are caused by underproduction of antibodies, other diseases may occur in which the immune system cannot produce effective amounts of protective antibodies to prevent infections or invasion. Infectious disease caused by bacteria or viruses, including the AIDS virus, and cancer represent conditions in which an antibody stimulatory drug could have significant therapeutic effects. Indeed, the major attempts to control AIDS infection by an AIDS vaccine are targeted towards stimulating antibodies against the AIDS virus. 5 Thus, peptides capable of stimulating B cell activity, or T. cell- activity mediating B cell maturation, may be used trx stϋnulate the body's natural antibody synthesizing machinery_/ in response to an acquired infection or a vaccine and would represent a useful therapeutic or 10 prophylactic tool.

Summary Of The Invention

The present invention is directed to methods and pharmaceutical compositions useful in the treatment of immunoinflammatory disease. Such diseases include

15 autoimmune disease, including demyelinating autoimmune neurologic diseases such as multiple sclerosis and postviral encephalomyelitis, autoimmune arthritic and joint-inflam atory diseases including rheumatoid arthritis and psoriatic arthritis, systemic lupus erythematosus and

2.0 juvenile-onset diabetes. Treatment of immunoinflammatory diseases such as organ transplantation rejection and graft-versus-host disease are also within the scope of the . veπtioπ:. rrπ addition, the present invention is directed to

257 methods and pharmaceutical compositions for the stimulation of immune responses including stimulation of TTcell sad B cell activity or proliferation, stimulation α£ .antibody synthesis and stimulation of immune responses against neoplastic or cancer cells.

30 The present methods include the therapeutic administration of pharmaceutical compositions comprising compounds referred to herein as Lys-Ser compounds, which includes in. particular compounds of the formula

R¹-Lys-Ser-R²

35: car a* pharmaceutically acceptable salt thereof, wherein R¹- is a- N*-substituent selected from R' - and R'CO-, and -R² is a carboxy-terminal substituentselected from -O ', -NHR* and -NR'₂, and where each R' is individually selected from hydrogen, branched and unbranched C^-C₃ lower alkyIs, C₂- C₈ alkenyls, C₂-C₈ alkynyls, C₆-C aryls, C₇-C alkaryls, C₇-C aralkyls and ₃-C cycloalkyls.

Brief Description Of The Drawings

FIG. 1 is a graph showing the neurologic deficit score of SJL/J mice immunized with spinal cord hymogenate in Complete Freund's Adjuvant and treated with saline or 1 mg Lys-Ser.

FIG. 2 is a graph showing the mean arthritis score (and S.E.M.) of DA rats treated every three days (days 0- 20, 10 mg; days 12-20, 1 mg) with Lys-Ser or control.

FIG. 3 is a graph showing the survival of NBR rat hearts transplanted into Lewis rat recipients and treated in vivo with Lys-Ser (5.0 mg/kg) or cyclosporin.

FIG. 4 is a graph showing the survival of NBR rat hearts transplanted into Lewis rat recipients and treated in vivo with Lys-Ser (1.0 mg/kg) or cyclosporin. FIG. 5 is a graph showing the time course (days) of survival of CBA/J bone marrow and T cell recipients treated with Lys-Ser or control.

FIG. 6 is a graph showing PHA-induced mitogenesis in rat splenocytes treated with intravenous Lys-Ser and control for 14 days.

FIG. 7 is a graph showing LPS-induced mitogenesis in rat splenocytes treated with intravenous Lys-Ser and control for 14 days.

Detailed Description Of The Invention Experiments undertaken with respect to the present invention have demonstrated the therapeutic utility of compounds related to the peptide Lys-Ser in treating or alleviating mammalian immunoinflammatory disease conditions. In addition, the immunoregulatory effects of such compounds in stimulating T cells and/or B cell activity or proliferation and in suppressing the growth of neoplastic cells have been demonstrated, as described herein and in parent Patent Application Serial No. 803,452. The present invention is directed to therapeutic uses of Lys-Ser compounds in treating immunoinflammatory diseases and in promoting other beneficial immune responses.

The therapeutic Lys-Ser compounds of the present invention include the dipeptide Lys-Ser and pharmaceutically acceptable derivatives and salts thereof. Appropriate derivatives may be formed by, for example, chemical substitution of Lys-Ser with acyl, alkyl and other substituents at the amino terminus of the molecule or by formation of esters or amides at the carboxy terminus. Thus, preferred Lys-Ser compounds are of the form R¹-Lys-Ser-R² wherein R¹- is a N*-substituent of the form R'- or R'CO- and -R_j is a carboxy-terminal substituent of the form -OR', -NHR^* or -NR'_g, where each R^* is individually hydrogen or an unbranched or branched C..-C₈ lower alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₆-C . aryl, C₇- C_χ4 alkaryl, C₇-C₁₄ aralkyl or ~--~ _Λ cycloalkyl.

Pharmaceutically acceptable acid or base addition salts of the foregoing compounds are also within the scope of the invention, and may include acid addition salts formed with one or more equivalents of an inorganic or organic acid (such as hydrochloric acid) or base addition salts formed with one or more equivalents of a desired base (such as a metal hydroxide base, a metal carbonate or bicarbonate base, or an amine base such as triethylamine or triethanolamine) . The hydrochloride salt and free base forms of the peptides are especially preferred.

Particularly preferred in the methods of the present invention is the peptide Lys-Ser. Highly preferred substituted Lys-Ser compounds include J^-acyl and alkyl derivatives formed from lower acyl or alkyl substituents, including most preferably N*-acetyl and ^-methyl derivatives, as well as carboxy-terminal esters and unsubstituted or primary amides formed from lower acyl substituents or unsubstituted amino or lower primary amino substituents, including most preferably methyl esters and -NH₂ amides. 5 The Lys-Ser compounds of the invention may be prepared using methods now standard in the chemical arts, and such procedures will not be repeated here. Reference may be had to the parent application to this application. Serial No. 803,452, to U.S. Patent No. 4,686,282, and to

10* the references cited therein, which are incorporated here by reference. Classical solution phase peptide synthesis, followed by conventional synthesis of appropriate derivatives, is specifically contemplated in the practice of the invention.

15 Useful pharmaceutical carriers for the medicinal formulation of the present peptides can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, powders, enterically coated or other protected formulations (such as by binding on ion

20) exchange resins or other carriers, or packaging in lipid protein vesicles) , sustained release formulations solutions (e.g., ophthalmic drops), suspensions, elixirs, aerosols, and the like. The carrier can be selected from the various oils including those of petroleum, animal,

25 vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic) for injectable solutions. Suitable pharmaceutical excipients

30⁾ include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The

~~ compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulation are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will, in any event, contain an effective amount of the active Lys-Ser compound and, generally, a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the host. A preferred dry form of the peptides is the stable, bulk lyophilized form of the free base (stored at 2°-8°C with desiccant) , and a preferred solution form is a phosphate buffered aqueous solution (stored at 2°-8°C) .

To be effective for the prevention or treatment of immunoinflammatory or other conditions it is important that the therapeutic agents be relatively non-toxic, non- antigenic and nσn-irritating at the levels in actual use.

In the practice of the methods of the present invention, an effective amount of a Lys-Ser compound, including salts, or a pharmaceutical composition containing the same, as discussed above, is administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the present invention or other pharmaceutical agents such as antihistamines, corticosteroids, and the like. These compounds or compositions can thus be administered orally, sublingually, topically (e.g., on the skin or in the eyes), parentally (e.g., intramuscularly, intravenously, subcutaneously or intradermally) , or by inhalation, and in the form of either solid, liquid or gaseous dosage including tablets, suspensions, and aerosols, as discussed in more detail above. The administration can be conducted in single unit dosage form with continuous therapy or in single dose therapy ad libitum.

In one preferred embodiment, the method of the present invention is practiced when the relief of symptoms is specifically required or perhaps imminent; in -another preferred embodiment, the^**methOT*^l-|&ereof is effeβlSjvely practiced as continuous or prophylactic treatment.

The appropriate dosage to be administered to a subject to achieve therapeutic reduction of an immunoinflammatory condition may vary depending on factors such as the mode and frequency of administration, the species being treated and the disease condition being treated. The examples given herein reflect representative doses shown to achieve beneficial activity, although it is contemplated that significant variations in the dosages may be utilized to achieve optimum therapy. In general, a preferred dosage range is from about 0.1 to about 100 mg/kg of Lys-Ser compound, depending on variables such as those noted above. Particularly preferred for inhibition of immune responses leading to inflammation is the range from about 0.25 mg/kg to about 10 mg/kg. Stimulation of immune system activity, as for example stimulation of differentiation of T cells and B cells (including stimulation of suppressor T cells, which can suppress an immunoinflammatory response, and stimulation of B cells, which can promote therapeutic antibody synthesis) is generally achieved at higher doses of the peptides, as for example from about 10 mg/kg to about 100 mg/kg. As with immunosuppression treatment, doses outside of this range may be utilized to achieve immunostimulation depending on the variables encountered.

In Vivo Efficacy Of Lys-Ser In Animal Models Of Autoimmunity

A. Multiple Sclerosis

Multiple sclerosis (MS) is believed to be an 5 autoimmune disease in which the immune system attacks the central nervous system (CNS) . While the precise cause of MS is unknown, genetic factors and possibly infection by certain viruses during childhood are postulated to cause the immune system, particularly T cells, to recognize O portions of the CNS as foreign, which leads to CNS injury and destruction. The autoimmune attack in MS is directed towards the myelin sheath of CNS cells which results in myelin destruction—hence the term "demyelinating neurologic disease" for MS and related conditions. 5 MS is a disease of young people, usually afflicting patients from 25 to 35 years old. The National Multiple Sclerosis Society estimates that approximately 250,000 people in the United States have MS with 10,000 new cases being diagnosed each year. MS produces muscle weakness, 0 paralysis, disturbances of vision and balance and other changes related to abnormalities of nerve conduction (Scheinberg, L., et al., "Multiple Sclerosis: Experimental and Clinical Aspects," Annals of the New York Academy of Sciences 436, 1984) . E55 The current methods of treating MS are disappointing. No treatment has demonstrated the ability to alter significantly the progress of MS over many years. Certain anticancer agents have shown a limited ability to reduce the severity of MS attacks, but are unable to alter the 0 ultimate disease course and have unacceptable levels of toxicity. Very recent clinical studies with the synthetic immunoregulatory protein, Copolymer-1, in patients with exacerbating-remitting MS have provided an encouraging indication that immunoregulatory substances may show 5 efficacy in MS therapy (Bornstein, M.B. et al., "A Pilot Trial of COP 1 in Exacerbating-Remitting Multiple Sclerosis," N. Enα . J. Med. 317:408, 1987). A combination of viral and immunologic factors have long been suspected in the etiology of MS (Lisak, R.P., "Multiple Sclerosis: Evidence for Immunopathogenesis," J. Neurol. 30(2) :99-105, 1980). Although there is compelling epidemiologic evidence to implicate an infectious agent as the cause of MS, no organism has been conclusively linked to the disorder (Kurtzke, J.F., et al., "Mortality and Migration in Multiple Sclerosis," Neurology 21:1186-97, 1971) . Considerable evidence has accumulated which supports the hypothesis that MS is caused by an autoimmune process. In 1948, Kabat and his colleagues demonstrated that the concentration of gamma globulin (IgG) in cerebrospinal fluid was elevated in patients with MS. While this observation has been confirmed and implies the presence of an ongoing immune-related process in the CNS, its pathogenic significance remains unclear. Histologic examination of the primary CNS lesion characteristic of MS, the MS "plaque", reveals large numbers of immunologically activated T.cells, B cells and lipid-laden macrophages at the demyelination front, suggesting an immunologically mediated process (Hofman, F.M. , et al. , "Immunoregulatory Molecules and IL 2 Receptors Identified in Multiple Sclerosis Brain," J. Immunol. 136:3239-3245, 1986) .

The development of animal models of MS, especially experimental allergic encephalomyelitis (EAE) has generated considerable support for the concept that T cells may be pathogenic in MS. (Raine, C.S., "Biology of Disease: Analysis of Autoimmune Demyelination: Its Impact Upon Multiple Sclerosis," Laboratory Investigation 50:608, 1984; and Brown, A.M., et al., "Relapsing Experimental Allergic Encephalomyelitis in the SJL/J Mouse," Laboratory Investigation 45:278-284, 1981). Studies of cells from human MS patients also suggest that T cells are involved in the MS tissue-destructive process. The suggestion that the balance of helper and suppressor T cells is altered at the onset of an MS attack, while controversial, provides insight into the type of immune abnormality that may underlie MS (Thompson, A.J., et al., "Peripheral Blood T-Lymphocyte Changes in Multiple Sclerosis: A Marker of Disease Progression Rather Than of Relapse?" J. Neurol. Neurosurg. S Psvch. 49:905, 1985) . Recent studies of T cell involvement in MS have focused on the potential role of suppressor T cells in the pathogenesis of MS. Human CD8⁺ (suppressor/ cytotoxic) T cell lines derived from patients with progressive multiple sclerosis, for example, have been reported to exhibit defective suppressor cell function (Antel, J. et al. , "Defective Suppressor Cell Function Mediated by T8+ Cell Lines from Patients with Progressive Multiple Sclerosis," J. Immunology 137:3436-3439, 1986). Very recent studies suggest that patients with chronic progressive MS have a selective loss of the cells necessary for the development of suppressor T cells (CD4⁺, 2H2⁺ suppressor-inducer T cells) (Morimoto, C. et al. , "Selective Loss of the Suppressor Inducer T-cell Subset in Progressive Multiple Sclerosis," N. Engl. J. Med. 316:67, 1987) . These data suggest that the autoimmune attack in MS may be due, in part, to a relative deficit of functional suppressor T cells. The utility of the present compounds is thought to reside, in part, in an ability to suppress the inflammation mediated by T cells in diseases such as MS.

Lvs-Ser Efficacy in an Animal Model of Multiple Sclerosis To evaluate Lys-Ser in an EAE animal model of MS, mice were immunized with central nervous system (CNS) tissue preparations to simulate the immunizing event thought to occur in humans. Animals with EAE have clinical, histological and immunological findings which resemble those found in human MS. The SJL/J mouse strain was selected in which to produce the EAE model. The EAE which occurs in mice seems more closely to resemble human MS than EAE induced in different animal models. This model is particularly advantageous for the study of T cell regulating compounds since the disease may be transferred into healthy mice using T cells from mice with EAE, indicating that EAE in SJL/J mice is predominately mediated by sensitized T cells (Mokhtarian, F. , et al., "Adoptive Transfer of Myelin Basic Protein-sensitized T Cells Produce Chronic Relapsing

Demyelinating Disease in Mice," Nature 309:356-358, 1984) .

Experiments conducted in conjunction with the present invention have shown that mice treated subcutaneously with

Lys-Ser experience a dramatic reduction in the development of EAE without evidence of peptide-induced toxicity. Histologic analysis of peptide-treated and saline control mice demonstrated a substantial reduction of lesions in the peptide-treated mice indicating that the fundamental pathogenic autoimmune process was being suppressed by peptide treatment (Armstrong, R.M. et al.. Presentation to the Canadian Neurologic Society International Meeting, June 1985) .

Experiments using a modification of the above EAE protocol have confirmed that Lys-Ser treatment has a substantial ability to inhibit the development of EAE. These studies have also demonstrated that peptide-treated animals with EAE experience a substantial reduction in the rate of disease relapse.

No signs of Lys-Ser toxicity were observed in the EAE experiments. Immunological monitoring of mice receiving Lys-Ser did not reveal evidence of broad immunosuppression common to conventional cytotoxic immunosuppressants.

These EAE experiments indicate that the Lys-Ser compounds of the invention can suppress the T cell mediated autoimmune attack that is believed to mediate the SJL/J model without significantly compromising normal immune function. Example 1

Experimental Allergic Encephalomyelitis Protocol for

Determining Efficacy in Treating Multiple Sclerosis

Female SJL/J mice (8 to 10 weeks old) , purchased from _> The Jackson Laboratories (Bar Harbor, ME) , were housed in groups; of ten per 19"xl0"x8" polycarbonate cage and were all wed! to acclimate for two weeks before using in experiments. Mice were given food (Wayne F6 Rodent Blox, Wayne. Eet Food, Chicago, IL) and water (pH 2.7) ad libitum - and maintained on a 12 hour light/dark cycle at 70 to 72°F and 45 to 55% relative humidity. Mice that developed signs of EAE were separated from the general population and manually fed and watered daily. Moribund mice were sacrificed by C0₂ asphyxiation. Spinal cord homogenate (SCH) in Complete Freund's Adjuvant (CFA) was prepared once at the beginning of each experiment following a modified version of the method developed by Brown and McFarlin (Brown, A.M., et al., "Relapsing Experimental Allergic Encephalomyelitis in the SJL/J Mouse," Laboratory Investigation 45:278-284, 1981). SJL/J donors were sacrificed by C0₂ asphyxiation and the cerebral, and spinal white matter were aseptically coϋected_* Tissues were washed in saline then were hramogen±zed twice for three minutes at 16,000 rpm with an : 0mni_mixe3r (Omni Corp., Waterbury, CT) in 1.0 ml phosphate buffered; saline per brain (PBS; 0.01 M phosphate, 0.15 M NaCH;; pH 7.4). The spinal cord homogenate mixture was lyophilized to dryness for 24 hours, and yielded an average of 35 mg per donor. A small sample of lyophilized, reconstituted tissue was streaked on blood agar (5% sheep blood; Re el, Lenexa, KS) to ensure that only sterile adjuvant was used in experiments. An equal volume_^ofPBS, containing 13.33 mg sterile SCH and 1.33 mg killed,. Mycobacterium tuberculosis (M. tb. : H37Ra; Difco Laboratories, Detroit, MI) , was combined with an equal volume incomplete Freund's adjuvant (Difco) to a final volume of 25 to 35 ml and were homogenized on ice twice for three minutes at 16,000 rp . The stable emulsion was stored at 4°C and used within two weeks of preparation.

Prior to immunization, SCH:CFA was loaded into 1.0 ml tuberculin syringes (Becton Dickinson, Rutherford, NJ) fitted with 21 g x 1" needles (Becton Dickinson) . SLJ/J mice (10 to 14 weeks old) were randomly distributed into experimental groups. Mice were inoculated subcutaneously at three dorsal sites with a total of 0.3 ml SCH:CFA (containing 1.0 mg SCH and 0.1 mg of M. tb.) on days 0 and 7. Preliminary experiments were performed to determine the optimal amount of M. tb. to produce EAE. For different M. tb. batches tested from 0.01 to 0.5 mg/mouse, the optimal amount varied from 0.1 to 0.3 mg per mouse. Mice were individually identified by ear punch.

The appropriate amount of Lys-Ser in 0.2 ml PBS

(sterile, pyrogen-free, 280 to 320 mOsm, pH 7.2 to 7.4) was injected subcutaneously using a 1.0 ml insulin syringe with a 27 g x 1/2" needle (Terumo Medical Corporation, Elkton, MD) . Since mice were treated daily, the injection was rotated over 7 consecutive dorsal sites to minimize trauma.

Animals were observed for neurologic deficit three times weekly beginning at the first sign of EAE, usually on day 14. The neurologic deficit score assigned to each mouse was based on the following classic ascending progression of signs characteristic of EAE: 0 — no deficits, 1 = flaccid tail, 2 = hind limb weakness, 3 = partial hind limb paralysis, 4 = paraplegia, 5 = quadriplegia, moribund. Clinical observations and treatments were assessed by research assistants who were not informed of the identity of the experimental groups until the end of the clinical observation period of each study. Figure 1 shows the neurological deficit scores of the SJL/J mice. Like the majority of human MS cases, the disease severity in this model waxes and wanes during the disease course. Mice receiving only saline injections two to three times per week exhibit high peaks and troughs of disease severity. By contrast, mice receiving Lys-Ser demonstrated very mild disease initially and were clinically disease-free by the study's end. Microscope analysis of brain sections from both saline and peptide- treated mice showed that one hundred percent of saline- treated mice demonstrated substantial numbers of visible lesions, representing physical brain destruction, in all portions of the CNS examined. By contrast, 60% of the peptide-treated mice were lesion-free while the remaining 40% had few, scattered lesions, confirming the clinically- elicited observations.

B. Rheumatoid Arthritis Rheumatoid arthritis (RA) is a chronic, systemic disease in which abnormalities of immune regulation cause inflammation and tissue destruction in joints and in other organs. The etiology of RA is unclear. Certain viruses, notably the Epstein Barr Virus (EBV) , have been implicated as pathogenic agents that may trigger the long-lasting immunoregulatory defects which characterize RA (Decker, J.L. (moderator) , "Rheumatoid Arthritis: Evolving Concept of Pathogenesis and Treatment," NIH Conference, Ann. Inter. Med. 101:810, 1984; and Depper, J.M., et al., "Epstein-Barr Virus: Its Relationship to the Pathogenesis of Rheumatoid Arthritis," Arthritis Rheum. 24:755, 1981).

Whatever the proximate cause, affected joints in RA are characterized by the presence of rheumatoid synovial tissue "pannus", an aggregation of activated and proliferating T cells, B cells and macrophages in a matrix of newly synthesized connective tissue. The inflammatory substances associated with pannus cells substantially contribute to the ultimate joint destruction which occurs over many years. RA is also characterized by an abnormally high synthesis of certain antibodies, termed "rheumatoid factors", directed against other immunoglobulin. These immune complexes activate many of the pathways that directly produce inflammation and cell destruction (Bromley, M. , et al., "Histopathology of the Rheumatoid Lesion: Identification of Cell Type at Sites of Cartilage Erosion," Arthritis Rheum. 27:857, 1984).

The two broad classes of therapeutic agents used to treat RA are the antiinflammatory drugs and the disease- modifying or anti-rheumatoid compounds. The antiinflammatory agents include the aspirin-like non- steroidal antiinflammatory drugs (NSAID) and steroids. The former provide primarily symptomatic relief by reducing the production of inflammatory mediators while having minimal effect on disease progression. Steroids can provide effective treatment of RA but are limited in their use by the severity of their side effects during chronic administration. The disease-modifying agents (DMARD) , including gold salts, D-penicillamine, and immunosuppressive and cytotoxic agents such as azathioprine, cyclophosphamide, methotrexate and lymphoid irradiation all suffer from variable efficacy and potentially severe side effects that limit their usefulness to the most serious cases not responding to palliative agents (Iannuzzi, L. , et al., "Does Drug Therapy Slow Radiographic Deterioration in Rheumatoid Arthritis?", N. Engl. J. Med. 309:1023, 1983).

Lvs-Ser Efficacy in an Animal Model of Rheumatoid Arthritis

Adjuvant-induced arthritis is an animal model of human rheumatoid arthritis in which immunologically- mediated inflammation is induced by the injection of killed bacteria in an oil emulsion ("adjuvant") . When adjuvant is injected into animals, a systemic immune response results in which both T cells, B cells and macrophages produce inflammation that particularly localizes to the joints (Wood, F.D., et al., "Capacity of Mycobacterial Wax D and Its Subfractions to Induce Adjuvant Arthritis in Rats," Int. Arch. Allergy Appl. Immunol. 35:456, 1969).

Lys-Ser was tested to evaluate its efficacy in adjuvant-induced arthritis in DA rats. Rats were given peptide by subcutaneous injection every three days only during the early stages of arthritis development. Peptide treatment was stopped after day 20 at which time the inflammatory process was continuing to develop. The peptide significantly inhibited arthritis in the limbs of afflicted rats when compared to saline-injection controls. Suppression of disease occurred in spite of the fact that peptide treatment was intermittent and was not continued throughout the evaluation period. The rats tolerated Lys- Ser treatment well and did not display observable toxicity.

Example 2

Adjuvant Arthritis Protocols for Determining Efficacy in

Treating Rheumatoid Arthritis

Adjuvant was prepared by adding heat-killed Mycobacterium butyricum (20 mg) to each milliliter of Complete Freund's Adjuvant (Difco). The mixture was stirred overnight before being used to achieve a uniform suspension of the bacteria. To induce arthritis, DA rats were lightly anesthetized with ether. Each rat was given 0.1 ml (5.0 mg mycobacterium) of the adjuvant preparation at the base of the tail.

The system described by Trentham, et al. (J. Exp. Med. 146:857, 1977) was used for judging arthritic responses. Based on a scale of 0 to 4 for each paw, the assessment was: 0, no response; 1, slight edema of the digital joints; 2, edema of the digital joints and footpad; 3, gross edema of the entire footpad below the joint; 4, edema of the entire foot including joint. In the more severe responses, swelling of the tail and ear was recorded, but no additional score was ascribed for these clinical signs. The highest score achievable was 16. Readings began on the tenth day after the adjuvant injection and continued every other day for twenty more days. A micrometer was used to quantitate edema. For select animals, histological and radiological evaluations was performed. Animals were sacrificed (etherization) on day after adjuvant injection. Blood was collected from selected rats by intracardiaσ bleeding of ether anesthetized animals. Serum was collected from clotted blood and stored at -20°C for further analysis. Two treatment protocols were followed:

(1) "Prophylactic" - Drugs administered every third days on days 0-20.

(2) "Therapeutic" - Drugs administered every third day on days 12-20. The control was the vehicle used to deliver peptide, isotonic saline, given according to either the "prophylactic" or "therapeutic" regimens as appropriate.

As shown in FIG. 2, Lys-Ser significantly inhibited arthritis in the limbs of afflicted rats when compared to saline-injection controls. Suppression of disease occurred in spite of the fact that peptide treatment was intermittent and was not continued throughout the evaluation period. The higher-dose (10 mg) peptide administration was given according to a prophylactic regimen, while the low-dose (1 mg) administration was given only four times after the disease-induced inflammation was evident according to a therapeutic regimen. In both cases the rats tolerated the treatment well and did not display observable toxicity.

C. Organ Transplantation Rejection and Graft-vs.- Host Disease

The immunological basis of transplanted organ rejection is very well understood and is essentially identical in animals and humans. Animal models developed to test the efficacy of potential antitransplantation rejection agents attempt precisely to duplicate the surgical procedures used in humans and have provided significant insight into the immunological basis of the rejection process.

Surgical transplantation of organs or cells between 5 genetically nonidentical individuals results in a cytotoxic immune response directed against the organ. Such rejection is primarily mediated by T cells which recognize histocompatibility molecules on the organ's cells. Treatments which destroy or substantially suppress

10) T cells can prevent the transplanted organ from being rejected (Bach, F.H., et al., "Current Concepts: Immunology Transplantation Immunology," N.Encrl. J. Med. 307:489, 1987).

Conventional cytotoxic immunosuppressive drugs such

15 as azathioprine, cyclophosphamide and prednisone produce the broad immunosuppression necessary to suppress T cells effectively, but substantially suppress normal immune responses as well. The relatively new agent, cyclosporin A, represents a great improvement in the selectivity of

20 the T cell-directed immunosuppression achieved. This agent, however, exhibits toxicity to both the liver and kidneys and in one to two percent of patients produces lymphoma (Kahan, B.D., et al., "Proceeding of the First International Congress on Cyclosporine," Transplantation

ZZ Proceedings XV No. 4, Supplements 1 & 2: Dec. 1983).

Lys-Ser Efficacy in Organ Transplantation Rejection and Graf -vs.-Host Disease

To evaluate the efficacy of Lys-Ser in prolonging the life of transplanted organs, hearts were transplanted into 30 genetically non-identical rats. Lys-Ser was administered by daily subcutaneous injection during the first ten post¬ operative days following which peptide treatment was stopped.

Lys-Ser was shown substantially to reduce the rate at

35 which transplanted hearts were rejected when compared to saline-injected control animals despite the fact that peptide was administered only during the first ten post¬ operative days. In addition, suppression of graft-vs.- host disease in a mouse model of bone marrow transplantation has been demonstrated. Peptide-treated animals displayed no signs of toxicity throughout these experiments.

Example 3 cardiac Transplantation Protocol for Determining Efficacy in Treating Organ Transplantation Rejection The heterotopic heart transplant model developed by Sun Lee, M.D. ("A Simplified Model for Heterotopic Rat Heart Transplantation," Nature 33:438-42, 1982) was used for introducing allogeneic hearts into recipient rats. This transplantation model is a relatively simple surgical procedure that requires a single end-to-side anastomosis, donor aorta to recipient dorsal aorta, and short ischemic times of approximately 30 minutes. Briefly, the donor was anesthetized using ethyl ether. The donor heart was removed by opening the thoracic cavity of the donor, and ligating and cutting the right and left superior vena cava. The aorta was then cut at the level of the innominate artery and the heart perfused with saline via the inferior vena cava. The inferior vena cava was then ligated and cut, the trachea cut and the heart and lung removed.

The recipient was anesthetized using ethyl ether and the abdomen entered through a midline incision. The infrarenal aorta was then isolated and clamped. An aortotomy was performed and an end-to-side anastomosis performed using a 9-0 nylon suture. The clamp was then released and donor heart contractions commenced spontaneously. After the donor heart resumed regular contractions, all of the lung lobes were ligated and removed, except the upper right lobe which provided complete circulation for the right heart. The recipient rats were treated by subcutaneous injection of Lys-Ser at dosages ranging from 0.2 mg/kg to

5.0 mg/kg or intramuscular injection with 15 mg/kg cyclosporin every day for ten days beginning on the day of

5 transplantation.

The resulting vascularized graft was monitored daily b palpation through the abdominal wall of the recipient. After a three day period, to ensure that the cessation of heart contraction was not due to a surgical etiology, the

ID- donor heart was considered rejected when contractions were no longer palpable, or electrical activity could no longer he. detected by EKG.

All rats used in these studies were inbred strains purchased from the Charles River Laboratory in Stone

15 Ridge, New York, or Trudeau Institute, Saranac Lake, New York facilities. The strain combination used for the heart transplant model was NBR (RT1^1/1) donors and Lewis (RT1^1/1) recipients, which resulted in rejection of the donor heart by untreated recipients in 20.7 ± 6.3 days.

2D Figure 3 shows the survival of the NBR rat hearts after transplantation into the Lewis rat recipients. Despite the fact that Lys-Ser administration (5.0 mg/kg) was continued for only ten days after transplantation, the life of the transplanted heart was prolonged as compared

255 tα a saline control. Similar results were obtained using as 1.0 mg/kg Lys-Ser administration (FIG. 4). A 0.2 mg/kg administration of Lys-Ser did not substantially prolong heart life compared to saline in the animals tested.

Example 4 30 Bone Marrow Transplantation Protocol for Determining Efficacy in Treating Graft-vs.-Host Disease

Suppression of GVHD was examined by using a bone marrow transplantation model using MHC compatible mice that differ at minor histocompatibility loci. Briefly,

35 B10.BR (H-2^k) bone marrow donors were sacrificed by cervical dislocation and the bone marrow removed from the right and left tibia and femur. Axillary superficial and inguinal lymph nodes were removed from the B10.BR mice and T cells isolated on nylon wool columns. Irradiated (800 rad) CBA/J (H-2^k) recipients were injected intravenously with 10⁷ bone marrow cells and 3 x 10⁶ isolated T cells from B10.BR donors. This transplantation model has been demonstrated to produce approximately 70% to 100% recipient mortality by day 32 after transplantation. Groups of ten bone marrow recipients each were treated for 40 days by daily subcutaneous injection of zero to 0.5 mg/kg Lys-Ser beginning on the day of transplantation. Transplant recipients were monitored daily for survival and body weight.

All mice used for GVHD studies were inbred strains purchased from Jackson Laboratories, Bar Harbor, Maine.

Figure 5 shows the percent survival of the tested mice at varying doses of Lys-Ser. At a 0.5 mg/kg dose, the survival rate was significantly greater as compared with a saline control.

EXAMPLE 5

Short-Ter Safety Studies of Lvs-Ser

To assess the short-term safety of Lys-Ser, five groups of Sprague-Dawley CD^R rats were used (12/sex/group) . The test material was administered daily for 7 or 14 days intravenously in the tail at dose levels of 1, 2, 10 and 100 mg/kg body weight. Phosphate buffered saline was injected into the control animals. After one week, 5 animals/sex/group were sacrificed while all remaining animals were sacrificed after two weeks. Selected organs were weighed and histopathologic evaluation of selected tissues was conducted on the control and high-dose animals. Physical observations, body weight and food consumption measurements were performed pre-test, at week 1 and on termination at week 2. Routine hematology and clinical chemistry analyses were performed on 5 animals/sex/group at week 1 and on termination at week 2. Test results indicated no mortality, remarkable clinical observations or treatment-related macroscopic or microscopic pathology. The treatment had no effect on body weight gains, organ weights, food consumption, hematologic or clinical chemistry parameters. Thus, the injection of 1 to 100 mg/kg of Lys-Ser i.v. into rats did not produce any short-term toxicity during this 14-day study.

Example 6 Acute Toxicity Studies

Acute toxicity studies were conducted using Lys-Ser to determine the lethal dose of the peptide from a single dose in 50% of the animals tested (LD₅₀) or to the limit of 5 g/kg body weight. Generally, if the LD₅₀ is determined to be greater than 5 g/kg body weight, then no additional acute toxicity testing is necessary. In the acute studies listed below, the highest possible dose of drug was used given the physiological limitations on route of

• administration. Each animal was administered a single dose of Lys-Ser and observed for 14 days for signs of pharmacologic and toxicologic effects. Body weights were recorded pretest, on Day 7 and on Day 14. The animals were sacrificed and gross postmortem examinations were performed on Day 14.

Table 1 Acute Toxicology Studies

Ad st ion Survived/Total Len th LD.

The results from the studies indicate that the acute intraperitoneal lethal dose (LD₅₀) of Lys-Ser in mice is greater than 5 grams per kilogram and in rats is greater than 2 grams per kilogram. The acute subcutaneous LD₅₀ in mice is greater than 5 grams per kilogram. The acute intravenous LD₅₀ in mice is greater than 1.6 grams per kilogram and in rats and acute intravenous LD₅₀ is greater than 1.0 gram per kilogram.

Immunomodulatory Effects Of Lys-Ser compounds

In vitro and ex vivo tests of Lys-Ser and related compounds have demonstrated that the compounds have a primary activity of suppressing T cell function, while in addition, at high doses, Lys-Ser appears to stimulate T cell function. Lys-Ser appears to achieve therapeutic efficacy by regulating the response of immunoactive cells in a manner similar to normal immunoregulatory "circuits".

Since the compound is identical in structure to a small portion of the IgG immunoregulatory molecule always present in the body, it is likely that its activity resembles one of the normal immunoregulatory functions of IgG or one of its proteolytically-derived metabolites.

Such activity may be achieved by binding to cellular receptors which directly trigger regulatory activities, hence yielding an immunomodulatory effect of, for example. stimulating or suppressing an overall immune response (depending on the particular cellular receptor bound) . In vitro studies of murine and human cells indicate that the compound can bind to IgG Fc receptors on lymphocytes and other leukocytes.

It is perhaps more likely that the functional Lys-Ser receptor is a molecule belonging to the molecular family from which both Fc receptors and immunoglobulin are derived — the immunoglobulin superfamily (Hahn, G.S., "Immunoglobulin-Derived Drugs," Nature 324 No. 6094:283- 284, 1986) . Molecules of the immunoglobulin superfamily include some of the most important regulatory molecules of the immune system and control virtually all aspects of immune responsiveness. This superfamily includes class 1 and 2 histocompatibility molecules, CD3 and Ti (members of the T cell antigen receptor complex) ; CD4 (helper- inducer T cell subset marker) ; CD8 (suppressor/cytotoxic T cell marker) ; Fc receptors for IgG, IgE, IgA and IgM and other molecules.

Example 7

Stimulatory Effect of Lvs-Ser on T cells and B cells

The immunomodulatory effects of Lys-Ser on CD rats given daily intravenous injections of varying doses of Lys-Ser for 14 days were assessed by measuring lipopolysaccharide (LPS) and phytohemagyluttin (PHA) induced mitogenesis in splenocytes. Splenocytes from treated Sprague-Dawley CD rats were cultured with LPS or PHA for three days at 37°C. DNA synthesis was measured by culturing the cells with 1 μCi tritiated thymidine for six hours. PHA proliferation (T cells) was inhibited at 1.0 and 2.0 mg/kg doses while a 100 mg/kg administration enhanced PHA proliferation by 133% in males and by 193% in females. See FIG.6. B lymphocyte proliferation (LPS) was enhanced (178-635%) in both sexes at a 100 mg/kg dose of the peptide. See FIG. 7.

Claims

Claims:

1. A pharmaceutical composition comprising a Lys- Ser compound of the formula

R¹-Lys-Ser-R² or a pharmaceutically acceptable salt thereof, wherein R¹- is a N^α-substituent selected from R'- and R'CO-, and -R² is a carboxy-terminal substituentselected from -OR', -NHR' and -NR'₂, and where each R' is individually selected from hydrogen, branched and unbranched C.,-C₈ lower alkyls, C₂- C₈ alkenyls, C₂-C₈ alkynyls, C₆-C₁₄ aryls, C₇-C₁₄ alkaryls, C₇-C_u aralkyls and C₃-C₁₄ cycloalkyls.

2. A pharmaceutical composition comprising Lys-Ser or a pharmaceutically acceptable salt thereof.

3. A method for treating mammalian immunoinflammatory disease comprising administering to a mammal a pharmaceutical composition comprising a Lys-Ser compound of the formula.

4. A method for treating mammalian immunoinflammatory disease comprising administering to a mammal a pharmaceutical composition comprising Lys-Ser or a pharmaceutically acceptable salt thereof.

5. The method of any of claims 3 or 4 wherein said disease is an autoimmune disease, graft-versus-host disease, systemic lupus erythematosus or juvenile-onset diabetes.

6. The method of any of claims 3, 4 or 5 wherein said Lys-Ser compound is administered in an amount of from about 0.1 mg/kg to about 100 mg/kg of body weight.

7. The method of claim 6 wherein said Lys-Ser compound is administered in an amount of from about 0.25 mg/kg to about 10 mg/kg of body weight.

8. The method of claim 3 wherein said Lys-Ser compound is administered according to a therapeutic regimen.

9. The method of claim 3 wherein said Lys-Ser 5 compound is administered according to a prophylactic regimen.

10. A pharmaceutical composition for stimulating antibody synthesis in a mammal comprising a Lys-Ser compound of the formula

10 R¹-Lys-Ser-R² or a pharmaceutically acceptable salt thereof, wherein R¹- is a N^Λ-substituent selected from R'- and R'CO-, and -R² is a carboxy-terminal substituentselected from -OR' , -NHR' and -NR'₂, and where each R* is individually selected from

15 hydrogen, branched and unbranched C,-C₈ lower alkyls, C₂- C₈ alkenyls, C₂-C₈ alkynyls, C₆-C₁₄ aryls, C₇-C₁ alkaryls, C₇-C₁₄ aralkyls and C₃-C₁₄ cycloalkyls.

11. A method of stimulating antibody synthesis in a mammal comprising administering to said mammal a

2Ω> pharmaceutical composition comprising a Lys-Ser compound αf the formula

R¹-Lys-Ser-R² or a pharmaceutically acceptable salt thereof, wherein R¹- is a I^-substituent selected from R'- and R'CO-, and -R²

Z5 is a carboxy-terminal substituentselected from -OR' , -NHR' and -NR*₂, and where each R* is individually selected from hydrogen, branched and unbranched C..-C₈ lower alkyls, C₂- C₈ alkenyls, C₂-C₈ alkynyls, C₆-C₁₄ aryls, C₇-C₁₄ alkaryls, C₇-C₁₄ aralkyls and C₃-C₁₄ cycloalkyls.

30 12. A pharmaceutical composition for stimulating a mammalian immune response against neoplastic cells comprising a Lys-Ser compound of the formula R¹-Lys-Ser-R² or a pharmaceutically acceptable salt thereof, wherein R¹- is a N^α-substituent selected from R'- and R'CO-, and -R² is a carboxy-terminal substituent selected from -OR', -NHR' and -NR'₂, and where each ¹ is individually selected from hydrogen, branched and unbranched C₁-C₈ lower alkyls, C₂- C₈ alkenyls, C₂-C₈ alkynyls, C₆-C₁₄ aryls, C₇-C₁₄ alkaryls, C₇-C₁₄ aralkyls and C_z-C cycloalkyls.

13. A method of stimulating a mammalian immune response against neoplastic cells comprising administering to a mammal a pharmaceutical composition comprising a Lys- Ser compound of the formula

R¹-Lys-Ser-R² or a pharmaceutically acceptable salt thereof, wherein R¹- is a N^α-substituent selected from R'- and R'CO-, and -R² is a carboxy-terminal substituentselected from -OR', -NHR' and -NR'₂, and where each R' is individually selected from hydrogen, branched and unbranched C,-C₈ lower alkyls, C₂- C₈ alkenyls, C₂-C₈ alkynyls, C₆-C₁₄ aryls, C₇-C₁₄ alkaryls, C₇-C₁₄ aralkyls and C₃-C₁₄ cycloalkyls.