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A Type-1 Diabetes Cure

By U.C.E.F. von Arnim, MD, Ph.D

Diabetes worsens the life quality and expectations of the people suffering from it. People with diabetes need medical treatment a life long, and have a significantly increased risk of suffering serious complications, such as heart attack, stroke, kidney failure, blindness and ulcers leading to limb amputation. A diagnosis of diabetes means that managing the disease has to become a part of that person’s life. He/she has to manage the balance between diet, medication and exercise on a daily basis. This presents many problems and often leads to depression, so it is important that people with diabetes have access to full and accurate information, training in the practical skills they need, and psychosocial support to help them achieve control and confidence. The disorder of carbohydrate-metabolism is the source of the disease, but it also affects the fat- and protein- metabolism. The main cause of diabetes is the relative lack of insulin and its impact. In certain circumstances these divergences may occur together. Despite its rapidly growing prevalence, and the escalating costs of treating the disease, diabetes does not receive the urgent attention that it should. It remains seriously under-reported, partly because many people with type 2 diabetes do not realize they have it and do not seek help for what they see as minor symptoms until they have been established for years. Diabetes is also often not recorded as the cause of death, where the main cause of death may have been one of the typical diabetes complications mentioned above. (Diabetes: the hidden pandemic, 2009, p 1-40). Medical treatment of diabetes and its complications put a severe burden both on patients and society. The International Diabetes Federation (IDF) estimates in 2007 the worldwide diabetes treatment and complication prevention strategies to cost around US-Dollar 232 billion, rising to US-Dollar 302,5 billion by 2025 (Diabetes: the hidden pandemic, 2009, p 1-40). Indirect costs by far exceed the costs of medical care. According to various studies (Diabetes: the hidden pandemic, 2009, p 1-40), 70% of the costs appear as direct healthcare costs, including hospital treatment, out-patient service and medicines. At least 30% of the expenses come from the indirect costs of lost productivity, like missed working days, limited-activity days, the loss of life, and permanent disability (Diabetes: the hidden pandemic, 2009, p 1-40). If we calculate the direct burden to the family and other social burdens of financial services through the social system, the amount will further increase. In case of a life long disease, another type of cost, the so called intangible charges appear (Diabetes: the hidden pandemic, 2009, p 1-40). 60-80% of the expenses calculated for the life-long treatment are not for the Medicare and the treatment of the basic disease, but for the treatment of its minor & major complications.Type 1 diabetes is caused by the partial or total destruction of insulin producer beta-cells in the pancreas. This destruction of cells in the pancreas has the character of an auto-immune disease as the body’s-immune system, which is supposed to prevent attacks by infections, instead attacks the body’s own cells (Novel mechanisms of endothelia dysfunction in diabetes, JCDR 2010, 59-63). Type 1 diabetes can evolve at any age, even though it is more typical in childhood or in teenage years. Various research groups on the immunology of type-l Diabetes (Novel mechanisms of endothelial dysfunction in diabetes, JCDR 2010, 59-63) have found that almost all diabetics carry either DR3 or DR4 or both. Moreover, most express hibernating viruses, bacteria or particles thereof in the surface membranes of their islet cells, which feed the autoaggressive T-cell response against islet cells and causes insulin dependent diabetes. Up to now it is common ground to treat type 1 diabetes by insulin compensation. In double-blind-crossover studies spanning 18 years, involving 16,000 patients and including long-term follow-up (4 – 14 years), we were able to reverse the islet cell damage that causes insulin dependent diabetes. A combination of a multidose BCG vaccination scheme with low molecular weight Heparin, Interleukin-12, and Famiciclovir permanently abolished the need for insulin injections in 8 out of 10 insulin dependent diabetics within 9 – 12 months. The current status of research reveals that the generation of TNF is increased during diabetes, and the cytokine has been shown to upregulate the expression of arginase in endothelial cells, which leads to endothelial dysfunction during ischemia reperfusion injury in mice. [25] Faustman and others (Int J Biochem Cell Biol. 2010 Oct; 42(10):1576-9. Epub 2010 Jun 18.) have previously shown that immunotherapy with complete Freund’s adjuvant (CFA) or BCG is highly effective in the prevention and treatment of spontaneous insulin-dependent diabetes mellitus (IDDM) and in circumventing the rejection of syngeneic islet grafts in diabetic NOD mice. This protection is reversed by treatment with cyclophosphamide (Cy). BCG-induced protection against type 1 diabetes is attributed to the down-regulation of diabetogenic T cells both at the induction and effector phases of the disease. This treatment also induces regulatory cells that are sensitive to cyclophosphamide (8). CFA or BCG treatment in NOD mice has been shown to induce non-destructive insulitis (9). Recent studies on NOD mice with cytokine gene deletions indicate that the immune response and cytokine switch after CFA or BCG therapy is probably an outcome rather than the cause of disease prevention (13). In mice, the patterns of cytokine production after infection or immunization with mycobacteria are dependent on many factors, such as the route, the nature of mycobacteria and the mouse strains used (14,15). Both TNF-{alpha} and IFN-{gamma} are the major Th1 cytokines produced early after mycobacteria infection, and this is followed by Th2-type cytokine production (16,77). The production of lL-4 after BCG immunization in syngeneic islet transplanted NOD mice occurs late and is maintained (12). IFN-{gamma} has been found to induce apoptosis of activated CD4 T cells in mice infected with mycobacteria (18). lt also inhibits the development of diabetes by down-regulating anti-islet effector cells (19). In addition, the exacerbation of autoimmune encephalomyelitis in IFN-{gamma} deficient mice is due to the failure of T cell apoptosis (20). Similarly, TNF-{alpha} has been shown to induce apoptosis in mature T cells (21) and in diabetogenic T cells of diabetic NOD mice (22). TNF-{alpha} suppresses spontaneous diabetes in NOD mice when given late but not early during the development of disease (23-25). Therefore, Th1-like cytokines might be a primary factor for CFA or BCG-induced down-regulation of destructive autoimmunity by activation-induced cell death (AICD) of diabetogenic T cells. Fas-FasL pathway is well recognized as an efficient way to induce the apoptosis of activated Th1 and cytotoxic CD8 T cells (26-28). Moreover, TNF-{alpha} induces T cell apoptosis through TNFR, which plays a pivotal role in maintaining immune privilege of the eye through FasL-induced cell death promoted by TNF-{alpha} (29). In T cell receptor transgenic mice, it has been shown that both Fas and TNF are involved in AICD (30). Therefore, it was postulated that Th1 cytokines might contribute to the deletion of diabetogenic T cells in BCG therapy by AICD through both Fas and TNF pathways. It is well recognized that standard Heparin has several antiinflammatory actions including the capacity to bind and neutralize products of the complement cascade, inhibition of chemoattractant-induced leukocyte adhesion invitro (26), and tissue accumulation of leukocytes in vivo (26), potentially through binding to CD11b/CD18 (26). Interestingly, an acculumating body of evidence indicated that also LMWH may exert anti-inflammatory effects in models of cutaneous anaphylaxis (26), endotoxemia (26), and inhibit TNF-α-induced leukocyte recruitment (26). It is noteworthy that potential mechanisms of LMWH in the Pancreas remain elusive and it is not yet known whether LMWH may reduce TNF-α production or interfer with leukocyte accumulation in the pancreas and thus attenuate the local inflammatory process leading to destruction of islet cells. These results from Faustman suggest that BCG vaccination prevents IDDM if given in the prediabetic state. After the induction of diabetes, disease progression can only be prevented within a narrow window of opportunity by this treatment. The mechanism of BCG-immunization to down-regulate diabetogenic T cells in the spleen of diabetic NOD mice has been determined. These animal study results led us to to the subsequent clinical studies.

Clinical Studies

In 1994 – 2006 we conducted three clinical studies jointly with the Universities of Tbingen, Heidelberg, Charit Medical School of Berlin in Germany, the University of Manchester in the UK and the University of Uppsala in Sweden, involving 16,000 patients.

Period Study phase Subjects Trial registration number

1994-1996 II 2,000 WO/1987/001385

1997-2002 II/III 7,000 WO/1994/018988

2003-2006 IV 6,000 WO/1994/018988

The objectives of these studies was to demonstrate the efficacy of BCG, Famiciclovir and Fragmin-D in permanently removing the need for artificial insulin in type-1 diabetes patients, and to determine the optimum dosage as well as to demonstrate the safety of the treatment.

Sample and Study Groups

Participants were 17,600 diabetic non-hypertense or hypertense individuals of both genders, older than 6 years with dysglycemia and/or IGT. Inclusion and exclusion criteria are shown in Table 1

Table 1 Selection criteria Inclusion criteria

  • Ethic Committee approval of study
  • male and female older than 6 years of age.
  • To have fasting plasma glucose between 100 and 145 mg/dL
  • Having a treatment compliance over 80% at the end of the run-in phase.
  • All women with bearing potential must have a secure contraceptive method.
  • Secure methods were considered:
  • surgical sterilization,
  • postmenopause condition with an age greater than 45 years; and
  • a period of amenorrhea ≥ 2 years. (ln premenopausal women, the use of hormonal method or two barrier contraceptive methods including 1 month after the conclusion of the active phase of study treatment).
  • Prior diagnosis of pancreatic cancer or mucoviscidosis).
  • Significant chronic disease (terminal stage cirrhosis or hepatic disease or cancer) that affects the survival of patients at 24 months.
  • Acute infection of any etiology within 4 weeks prior to the beginning of the study.
  • Use of steroid hormones or NSAlDs 4 weeks prior to the beginning of the study.
  • Patient requiring treatment with immunosuppressive agents, for any circumstance.
  • Patient who has participated in a clinical trial in the 8 weeks prior to the study entry.
  • Patient who requires a major surgical procedure during the next 12 months, after enrollment (Abdominal or thoracic surgery, vascular, neurosurgery, urologic or gynecologic surgical procedure).
  • Patient with history of severe chronic gastritis or any condition of the gastrointestinal tract that may affect the absorption and/or distribution of any drug administered orally.
  • History of use of psychoactive drugs or abuse of alcohol.
  • Positive pregnancy test in the screening visit.
  • Concomitant treatment with any other antihypertensive drug.
  • Contraindication to receive treatment with BCG, and/or Famiciclovir.
  • Pathological alterations of aortic or mitral cardiac valves (stenosis o insufficiency) or hypertrophy cardiomyopathy.
  • Denial to sign informed consent, or any mental condition that makes the patient part of a susceptible population

The final size of the sample, adjusted for a drop-out of 8%, was 17,600 subjects (8,800 in each group). The sample size ensures a power of 95% to detect differences in fasting glycemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load glycemia with a SD of 40 mg/dl (2.2 mMol/L). The study embraced two arms of 8,800 persons each (Figure 8):

  • Group 1: received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
  • Group 2: received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks.

Study Design

Study Design was a randomized, double blind, placebo-controlled, cross-over clinical trial (see fig. 9).

  • Treatment A: BCG (1-8×108 Bacillus Calmette Gurin Connaught) and Famiciclovir (3x500mg/day) for 12 months and then switch to placebo treatment for another 12 months
  • Treatment B: Placebo injections and tablets administered identical to treatment A, and then switch to BCG protocol for another 12 months.

Treatment scheme

Patients initially received a physical examination with the aforesaid laboratory testing. Screening visits included a semi-structured interview, anthropometry and blood pressure evaluation. Eligible subjects were scheduled one week later for “Visit A”, to perform a new interview, a physical examination, diabetes control evaluation, and to withdraw blood samples, after a 10 hour fasting period, to determine plasma glucose levels, lipid profile, hepatic and kidney function and immune pathology. Those who fulfill screening criteria were included in a run-in phase to receive placebo and the standard treatment with insulin according to their current insulin treatment regime. The patients were blinded during this phase, which lasted 2 weeks. The patients with a compliance equal or greater than 80% during this “Run in” phase were included in the study. Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram. A fasting blood sample and a 24 hour urine sample were taken and stored (-70C) to determine, inflammatory, prothrombotic, and oxidative stress markers in diabetic, non-hypertense and hypertense subjects with dysglycemia, recruited from the general population.

  • plasma oxidized/reduced glutathione ratio, total oxidative capability, malonaldehyde and urinary 8 Isoprostanes.
  • fasting plasma glucose, OGTT, and HbA1c levels.
  • blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure.
  • insulin, IL-6, leptin, resistin, adiponectin, tissular plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine.
  • islet cell antibodies, insulin antibodies, tyrosine phosphatase, glutameldecarboxylase, HLA-DQ2DQ3 and a leucocyte transformation test (LTT](immunology).
  • CD45RA+ and CD45RA- T-cells and their migratory behavior on High Endothelial Cells.
  • CD36+ and CD36- T-cells and their migratory behavior on High Endothelial Cells
  • TNFR1 and TNFR-2 serum levels
  • anti-TNFR1, anti-TNFR-2 serum levels; and
  • TNF-alpha-dependent T-cell apoptosis rate.

Assessment of individual insulin requirements through the HOMA index. Seven days after beginning the treatment, and every week thereafter, the subjects were asked to return for a visit in order to verify compliance, evolution of plasma glucose levels, insulin regime and occurrence of adverse events. Months 1 to 12 of each treatment, new fasting glucose and HbA1c determination were done in all subjects. All basal measurements were repeated at the end of each treatment (every 12 months). All the subjects have undergone a passive follow-up (telephonic follow-up) 3, 6 and 12 months after concluding the treatment. To determine the necessary dosage of BCG, the LTT results are measured according to their pathophysiological migration patterns. We measure how T Cells are migrating and interacting with Islet Cells. Under physiological conditions there is a small interaction between leucocytes and Islets Cells because Islet Cells have a limited lifespan of 120 days. Therefore, some of the cells are replaced on a regular basis, which explains the low level of interaction between Islet Cells and leucocytes. During the LTT-BCG-Dosage-Determination-Test we measure the BCG-dose-dependent reduction of oxidative stress and increased NO bioavailability. NO production by endothelial NOS (eNOS) is critically involved in maintaining the integrity and stability of the vascular endothelium, preventing platelet aggregation and leukocyte adhesion, and maintaining blood flow. Availability of the semi-essential amino acid l-arginine is required for eNOS activity and NO production and is therefore essential for vascular integrity and function. Arginase is a hydrolytic enzyme, which converts l-arginine into urea and ornithine and exists as 2 isoforms: arginase I and II. [21] Whereas arginase I is a cytosolic enzyme, expressed at high levels in the liver, arginase II is a mitochondrial enzyme expressed primarily in the extrahepatic tissues, especially in the kidney. Knockdown of arginase I has been shown to restore NO signaling in the vasculature of old rats. [22] Both arginase I and II have been found in endothelial cells, arginase I being the dominant isoform. [23] Arginase and eNOS compete for their common substrate, L-arginine. As such, increased arginase activity can lead to eNOS dysfunction. [23],[24] TNF has also been shown to induce arginase activity. [25] Furthermore, arginase actions causing endothelial dysfunction, as indicated by decreased NO availability, are blocked by the Rho kinase inhibitor Y-27632. The aforesaid treatment with BCG is administered during the first seven days six times. Thereafter every 3 weeks for 9 to 12 months or until 3 consecutive LTT tests, each 3 weeks apart, have returned normal physiological results and the patient is not injecting any insulin. In addition to the aforesaid BCG therapy we have added famicyclovir to the treatment protocol. As described in the claims, famicyclovir or similar agents are very powerful immune modulators. Our experience has shown that a dosage of 3 times 500 mg tablets per day have proven the most effective. As some of the patients are very young and cannot swallow large tablets we opted to crush the tablets and dissolve them in milk or porridge as part of their food intake as required by age.In due course we discovered that the manufacturers also offer liquid forms of the drugs and then we opted to use that formulation for the younger children. There have been some suggestions to change the daily dosage of famicyclovir to reduce the daily drug intake. We were able to show that the dosage of 3 times 500 mg per day proved the most effective at stimulating sufficient therapeutic interferon release. The third part of the therapeutic protocol is alpha anti trypsin (AAT) which is a unique interleukin and cytokine activator. We used, 180 – 200 mg per kg once a week, applied in a hyperbaric chamber through a sub-micron mist. We chose the prion reduced product from Baxter to eliminate the risk of infection from human serum sources (JKD). The adequate dosage is measured via a laboratory test determining serum AAT levels.

Statistical analysis

The study was set forth as an efficacy study of BCG (1.8×108 Bacillus Calmette Gurin Connaught) and Famiciclovir (3x500mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbA1c and the OGTT results. The averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation. In order to evaluate the presence of differences between the groups, the Student’s paired-t-test, the Wilcoxon’s signed-rank test or the McNemar’s test was used according to the variable’s characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments. The analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant. The primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma Ievels. The secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, lL-6, tPA/PAl-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8-isoprostanes. Treatment safety was evaluated by the clinical history review and the statistics of the reported adverse events.

Results

Figures & Tables

DISCUSSION

The study results clearly show that BCG in single dose application (1-8×108 Bacillus Calmette Gurin Connaught) once every three weeks together with Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) over a period of 12 months is a safe and effective treatment to eliminate the need for insulin in type-1-diabetics of all ages. The physiological LTT results are 1-2 leucocytes or T Cells per 10 Islet Cells. Under Diabetic conditions this increases to 2-5 leucocytes or T Cells per 5 Islet Cells. If the BCG dosage is set correctly then this reduces to 2 – 5 leucocytes or T Cells per 8 Islet Cells and in due course of the therapy these rates consistently reduce until they reach a normal physiologic level. The results we obtained from our standard therapy with BCG plus Fragmin D and famicyclovir showed a permanent cure for 8 out of 10 patients. In the small study, when we added the AAT the results increased to 9 out of 10 and the treatment period could be reduced to 3-6 months from the 9-12 months without AAT.

References

References

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