Objective
Our objective is to develop a lacritin replacement therapy for the prevention of recently diagnosed T1D. Initial studies were with the N-104 lacritin fragment that overlaps with patented LacripepTM. From our prior knowledge of the biological activity of lacritin, we invented a novel 'N-104-like' fragment known as 'RegenpepTM' that is equally effective as N-104 in islet cell culture experiments. Our objective in this proposal is to determine whether PEG-RegenpepTM blocks autoimmunity with equal efficacy in vivo as PEG-N-104. We know that subcutaneous injection of PEG-N-104 suppresses the formation of precursors of the cytotoxic T cells responsible killing insulin secreting beta-cells. Can PEG-RegenpepTM do the same and can we learn more about this mechanism? An excellent understanding of mechanism is essential for moving the drug into the clinic.
Background Rationale
To address the biological basis of autoimmune dry eye disease, we discovered and named the protein 'lacritin', a tear protein. Lacritin is important for the health of the surface of the eye. In autoimmune dry eye disease, the active form of lacritin is selectively deficient. We therefore created a drug as a lacritin replacement therapy. The drug, 'LacripepTM' has been tested as a topical therapeutic in autoimmune dry eye with success. Further trials are occurring this summer towards eventual FDA approval. Our diabetes project was initiated almost seven years ago when my lab was temporarily translocated for renovation to another building where a chance encounter initiated a collaboration with a group interested in T1D. The lab head is a transplant surgeon who transplants isolated human islets in severe T1D patients. Islets are not easy to acquire and are unstable after cadaveric isolation. When incubated with LacripepTM, stability was enhanced. Even better results were obtained with the lacritin fragment 'N-104', which is slightly shorter than LacripepTM. Other studies followed. N-104 was found to substantially enhance islet glucose-stimulated insulin secretion, to regenerate insulin-secreting beta-cells, to restore normal blood glucose levels when N-104 treated islets were transplanted into diabetic mice and to block the development of diabetes in mice when PEG-N-104 was injected under the skin. We stopped the injection experiment at ten months when among ten NOD mice receiving treatment, only one became diabetic. Further examination revealed that autoimmunity had been blocked. Lacritin is also in blood to benefit islets of the pancreas. However, in T1D, the active form of lacritin (as in autoimmune dry eye disease) is deficient. Taken together with our observations in T1D mouse models in mice, that N-104 blocks T1D autoimmunity and restores insulin-secreting beta-cells and enhances insulin secretion in the presence of high glucose, we wonder whether early introduction of N-104 replacement therapy might prevent the development of T1D in humans. No other drug does this. Attempts at prevention of T1D have largely focused on early immunomodulatory (e.g., FDA approved anti-CD3) or preclinical islet beta-cell regenerative approaches. Combining both approaches in a single, well tolerated therapeutic could be beneficial.
Description of Project
We introduce a 'lacritin' fragment to the type 1 diabetes (T1D) community as the first drug capable of both blocking autoimmunity and restoring beta-cells. Lacritin is a tear, plasma and cerebral spinal fluid protein discovered and named 24 years ago by the Laurie lab out of an unbiased biochemical screen to address the biological basis of autoimmune dry eye disease. In dry eye, the active, monomeric form of lacritin is selectively deficient - much as we have found in blood of adult patients with T1D. Deficiency appears to be disease causative not only for the eye, but also likely for pancreatic islets. Through lacritin's multiple functions, it is essential for health and is regenerative. All functions are contained in the C-terminal lacritin fragment 'N-104' that overlaps with 'LacripepTM', the topical drug that we developed for autoimmune dry eye and have successfully tested in one clinical trial with more planned. In this proposal, we introduce the N-104 inspired and recently patented drug 'RegenpepTM'. (N-104 is not patentable because of its overlap with patented LacripepTM; N-104 is more active on islets than LacripepTM). RegenpepTM enhances glucose-stimulated insulin secretion by isolated human and mouse islets. Further, RegenpepTM treated human islets minimally transplanted into T1D mice restore normoglycemia. The aims of this proposal are to test: 1) whether RegenpepTM, like N-104, can block autoimmunity in the NOD mouse T1D model and explore how N-104 and RegenpepTM may change the microenvironment of the pancreatic draining lymph nodes from which precursors of cytotoxic, beta-cell killing T-cells arise; and 2) to confirm that N-104 and likely RegenpepTM suppress such T-cell precursors, determine how this occurs and whether expression of a cytokine is involved. The goal is a RegenpepTM treatment regimen for early stage T1D as a replacement therapy for deficient lacritin (much as is LacripepTM for dry eye). Data from this proposed study serve as the foundation for FDA investigative new drug (IND) preclinical studies towards human clinical trials. For in vivo drug delivery studies, FDA approved polyethylene glycol is added to N-104 (PEG-N-104) and RegenpepTM (PEG-RegenpepTM).
Anticipated Outcome
We anticipate that RegenpepTM will perform equally well as N-104. We will learn how N-104 (and likely RegenpepTM) reverses T1D in mice. Related lacritin fragment, LacripepTM has already been tested as a topical for dry eye in humans. Rapid efficacy was observed as well as excellent toleration.
Relevance to T1D
We introduce 'lacritin' to the type 1 diabetes (T1D) community as the first drug capable of both blocking autoimmunity and restoring beta-cells. Lacritin is a tear, plasma and cerebral spinal fluid protein discovered and named 24 years ago by the Laurie lab out of an unbiased biochemical screen to address the biological basis of autoimmune dry eye disease. In dry eye, the active, monomeric form of lacritin is selectively deficient - much as we have found in blood of adult patients with T1D. Deficiency is disease causative not only for the eye, but also likely for pancreatic islets. Through lacritin's multiple functions, it is essential for health and is regenerative. All functions are contained in the C-terminal lacritin fragment 'N-104' that overlaps with 'LacripepTM', the topical drug that we developed for autoimmune dry eye and have successfully tested in one clinical trial with more planned. In this proposal, we introduce the N-104 inspired and recently patented drug 'RegenpepTM'. (N-104 is not patentable because of it overlap with patented LacripepTM; N-104 is more active on islets than LacripepTM). RegenpepTM enhances glucose-stimulated insulin secretion by isolated human and mouse islets. Further, RegenpepTM treated human islets minimally transplanted into T1D mice restore normoglycemia. The aims of this proposal are to test: 1) whether RegenpepTM, like N-104, can block autoimmunity in the NOD mouse T1D model and explore how N-104 and RegenpepTM may change the microenvironment of the pancreatic draining lymph nodes from which precursors of cytotoxic, beta-cell killing T-cells arise; and 2) to confirm that N-104 and likely RegenpepTM suppress such T-cell precursors, and to determine how this occurs and whether expression of a cytokine is involved. The goal is a RegenpepTM treatment regimen for early stage T1D as a replacement therapy for lacritin in T1D blood (much as is LacripepTM for seletive lacritin deficiency in dry eye). Data from this proposed study serve as the foundation for FDA investigative new drug (IND) preclinical studies towards human clinical trials.