U.S. patent application number 14/887427 was filed with the patent office on 2016-02-04 for method for reversing recent-onset type 1 diabetes (t1d) by administering substance p (sp).
The applicant listed for this patent is Hans-Michael DOSCH. Invention is credited to Hans-Michael DOSCH.
Application Number | 20160030504 14/887427 |
Document ID | / |
Family ID | 52777431 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160030504 |
Kind Code |
A1 |
DOSCH; Hans-Michael |
February 4, 2016 |
METHOD FOR REVERSING RECENT-ONSET TYPE 1 DIABETES (T1D) BY
ADMINISTERING SUBSTANCE P (sP)
Abstract
Described herein is a treatment comprising the following step:
(a) injecting a therapeutically effective amount of a
pharmaceutical composition into the celiac artery of an individual,
wherein the pharmaceutical composition reverses recent onset Type 1
Diabetes (T1D). Also described is a method for identifying an
individual who will be responsive to this treatment. In addition
there is described a device containing the pharmaceutical
composition for injecting the pharmaceutical composition into the
celiac artery.
Inventors: |
DOSCH; Hans-Michael;
(Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DOSCH; Hans-Michael |
Toronto |
|
CA |
|
|
Family ID: |
52777431 |
Appl. No.: |
14/887427 |
Filed: |
October 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14244990 |
Apr 4, 2014 |
9192647 |
|
|
14887427 |
|
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|
61886804 |
Oct 4, 2013 |
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Current U.S.
Class: |
514/6.7 ;
514/7.3 |
Current CPC
Class: |
A61P 3/10 20180101; C12Q
1/6883 20130101; C12Q 2600/156 20130101; A61K 38/046 20130101; A61P
3/00 20180101; A61K 9/0019 20130101; C12Q 2600/106 20130101 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method comprising the following step: (a) injecting a
therapeutically effective amount of a pharmaceutical composition
into the celiac artery of an individual, wherein the pharmaceutical
composition at least partially reverses recent onset Type 1
Diabetes (T1D).
2. The method of claim 1, wherein the therapeutically effective
amount of a pharmaceutical composition is injected into the celiac
artery of an individual as a single dose of the pharmaceutical
composition.
3. The method of claim 1, wherein the pharmaceutical composition
completely reverses recent onset T1D.
4. The method of claim 1, wherein the pharmaceutical composition
transiently for months to years reverses recent onset T1D.
5. The method of claim 1, wherein the pharmaceutical composition
aids storage, production and release of insulin by beta cells in a
pancreas.
6. The method of claim 1, wherein step (a) is conducted for at
least three minutes.
7. The method of claim 1, wherein the pharmaceutical composition
comprises a neuropeptide.
8. The method of claim 1, wherein the pharmaceutical composition
comprises substance P.
9. The method of claim 8, wherein substance P is dissolved in
saline in the pharmaceutical composition.
10. The method of claim 1, wherein the individual is diagnosed with
Type 1 Diabetes (T1D).
11. The method of claim 10, wherein the individual is diagnosed
with T1D based on DNA from a saliva sample obtained using a DNA
collection kit.
12. The method of claim 1, wherein the individual is a human.
13. The method of claim 1, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 10 nM/kg.
14. The method of claim 1, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 50 nM/kg.
15. The method of claim 1, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 100 nM/kg.
16. The method of claim 1, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 250 nM/kg.
17. A device for injecting a pharmaceutical composition into the
celiac artery of an individual, wherein the device contains a
therapeutically effective amount of the pharmaceutical composition,
and wherein the pharmaceutical composition at least partially
reverses recent onset Type 1 Diabetes (T1D.sub.[MG2]).
18. The device of claim 17, wherein the device comprises a catheter
and wherein the catheter contains the therapeutically effective
amount of the pharmaceutical composition.
19. The device of claim 17, wherein the pharmaceutical composition
completely reverses recent onset T1D.
20. The device of claim 17, wherein the pharmaceutical composition
transiently for months to years reverses recent onset T1D.
21. The device of claim 17, wherein the pharmaceutical composition
aids storage, production and release of insulin by beta cells in a
pancreas.
22. The device of claim 17, wherein the pharmaceutical composition
comprises a neuropeptide.
23. The device of claim 17, wherein the pharmaceutical composition
comprises substance P.
24. The device of claim 23, wherein substance P is dissolved in
saline in the pharmaceutical composition.
25. The device of claim 17, wherein the individual is diagnosed
with Type 1 Diabetes (T1D).
26. The device of claim 17, wherein the individual is a human.
27. The device of claim 17, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 10 nM/kg.
28. The device of claim 17, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 50 nM/kg.
29. The device of claim 17, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 100 nM/kg.
30. The device of claim 17, wherein the therapeutically effective
amount of the pharmaceutical composition is at least 250 nM/kg.
31. The device of claim 17, wherein the device is inserted into the
celiac artery of the individual.
32. A method comprising the following step: (a) identifying an
individual with recent onset Type 1 Diabetes (T1D) who will respond
positively to injecting a therapeutically effective amount of a
pharmaceutical composition into the celiac artery of the
individual, wherein the pharmaceutical composition at least
partially reverses recent onset Type 1 Diabetes (T1D) in the
individual.
33. The method of claim 32, wherein the individual in step (a) is
identified based on DNA in a saliva sample from the individual
obtained using a DNA collection kit.
Description
[0001] This application is a divisional of application Ser. No.
14/244,990, filed Apr. 4, 2014. This application also claims
benefit of priority to U.S. Provisional Patent Application No.
61/886,804 filed Oct. 4, 2013, which is incorporated herein by
reference in its entirety.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The present invention relates to neuropeptide therapy of
recent-onset Type 1 diabetes (T1D).
[0004] 2. Related Art
[0005] Current methods of treatment and management of Type 1
Diabetes (T1D) include insulin replacement therapy, pancreas
transplantation and islet cell transplantation. Insulin replacement
therapy is a challenging and uncomfortable lifelong process plagued
by side effects and it does not cure T1D. The clinical challenge is
the necessity to adjust insulin release with control of
minute-to-minute changing needs. Insulin replacement therapy as
practiced saves lives, but cannot prevent chronic succession of
hypo- and hyperglycemic events that ultimately degrade micro- and
macrovascular functionalities with broadly progressive tissue
damage and neuropathy.
SUMMARY
[0006] According to one broad aspect, the present invention
provides a method comprising following step: (a) injecting a
therapeutically effective amount of a pharmaceutical composition
into the celiac artery of an individual, wherein the pharmaceutical
composition at least partially reverses recent onset Type 1
Diabetes (T1D).
[0007] According to a second broad aspect, the present invention
provides a device for injecting a pharmaceutical composition into
the celiac artery of an individual, wherein the device contains a
therapeutically effective amount of the pharmaceutical composition,
and wherein the pharmaceutical composition at least partially
reverses recent onset Type 1 Diabetes (T1D).
[0008] According to a third broad aspect, the present invention
provides a method comprising the following step: (a) identifying an
individual with recent onset Type 1 Diabetes (T1D) who will respond
positively to injecting a therapeutically effective amount of a
pharmaceutical composition into the celiac artery of the
individual, wherein the pharmaceutical composition at least
partially reverses recent onset Type 1 Diabetes (T1D) in the
individual.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The accompanying drawings, which are incorporated herein and
constitute part of this specification, illustrate exemplary
embodiments of the invention, and, together with the general
description given above and the detailed description given below,
serve to explain the features of the invention.
[0010] FIG. 1 is a drawing showing a catheter injecting a
pharmaceutical composition into the celiac artery of an individual
according to one embodiment of the present invention.
[0011] FIG. 2 is a graph showing changes in blood glucose levels,
in recent onset diabetic mice, following intra-arterial injection
of the pancreas with substance P (sP).
[0012] FIG. 3 is a graph illustrating the CFSE T cell proliferation
assay inside the pancreatic lymph node of a transgenic, diabetic
mouse carrying a diabetogenic T cell receptor (TCR).
[0013] FIG. 4 is an image of a typical, no longer infiltrated islet
after recent onset Type 1 diabetes and intra-arterial pancreatic
substance P delivery.
[0014] FIG. 5 is an image showing vasodilated pancreatic tissue in
mice, following intra-arterial pancreas injection with substance P
plus Evans Blue dye marker (observe: pancreas-selective,
grayish-blue coloration).
[0015] FIG. 6 is a graph that illustrates alternative outcome
models of the overall study plan.
[0016] FIG. 7 shows a DNA collection kit that may be used in one
embodiment of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] Where the definition of terms departs from the commonly used
meaning of the term, applicant intends to utilize the definitions
provided below, unless specifically indicated.
[0018] For purposes of the present invention, a value, a
determination or a property is "based" on a particular value, data,
information, property, the satisfaction of a condition, or other
factor if that value, determination or property is derived by
performing a mathematical calculation or logical decision using
that value, property or other factor. For example, a determination
that an individual has Type 1 Diabetes (T1D) based on the DNA
obtained from a saliva sample from the individual
[0019] For purposes of the present invention, the term "catheter"
refers to a thin tube extruded from medical grade materials that
facilitates the access of the pancreas.
[0020] For purposes of the present invention, the term "individual"
refers to a mammal. For example, the term "individual" may refer to
a human individual.
[0021] For purposes of the present invention, the term
"neuropeptide" refers to a small protein-like molecule or peptide
released by sensory afferent neurons to communicate with their
innervated target tissue.
[0022] For purposes of the present invention, the term
"recent-onset Type 1 Diabetes (T1D)" refers to a clinical disease
onset up to 30 months prior reversal therapy.
[0023] For purposes of the present invention, the term "respond
positively to injecting a therapeutically effective amount of a
pharmaceutical composition into the celiac artery of the
individual" refers to an individual for whom this injection of the
pharmaceutical composition will at least partially reverse the
effect of recent onset Type 1 Diabetes (T1D) in the individual.
[0024] For purposes of the present invention, the term
"therapeutically effective amount" refers to an amount of substance
P administered to an individual that is effective in complete or at
least partially reversing recent-onset Type 1 Diabetes in the
individual.
[0025] For purposes of the present invention, the term "Type 1
Diabetes (T1D)" refers to a form of diabetes mellitus that results
from autoimmune destruction or de-differentiation of
insulin-producing beta cells of the pancreas.
DESCRIPTION
[0026] Transient receptor potential Vanilloid-1, a neuronal
Ca.sup.++-channel, TRPV1, is a major heat-sensing protein in
sensory afferent or "pain" nerves. When activated, these nerves
release very small biological mediators, neuropeptides, such as the
11-amino acid substance P. sP widens arteries and attracts many
elements of the immune system in the area where TRPV1 was
activated. It is known that TRPV1 produces a mediated heat
sensation. It is known that the burning sensation (generated in the
brain) and the local vasodilation/hyperemia (reddening) produced by
sP released by activated TRPV1 nerve endings results in the sensing
of a heat insult (.about.45.degree. C.) to the skin: the "ouch"
response learned by toddlers encountering something that is
"hot".
[0027] The sP/sP-receptor (also called NK1R-) pathway is
essentially universal, with multiple, general and tissue-specific
roles in diverse tissue/organ compartments. In working on the
present document, it is estimated from independent searches, that
there are more than 30,000 scientific articles that use, measure,
map sP and its cousin neuropeptide, CGRP in relation to various in
vivo or in vitro interactions and effects. The current NIH
trials.gov registration site lists nearly 2000 current clinical
trials when queried with "substance P".
[0028] sP acts as a neurotransmitter, and central neurotransmission
along the sP/NK1R pathway was more recently identified as critical
modifier in the pathophysiology of varied neuropsychiatric
conditions, anxiety, depression, fear, phobia, migraine and diverse
stress syndromes, well beyond its early recognized roles in acute
and chronic pain, analgesia, emesis--the list of processes
involving the sP/sP receptor axis in health and disease is still
growing (Haneda et al., 2007.sup.29; Michelgard et al.,
2007.sup.30).
[0029] There is a family of TRPV-like sensors, with TRPV1 the
founding and most widely expressed member (Dorfman et al.,
2010.sup.4). TRPV1 integrates multiple and diverse body bitmaps in
the brain. When you tickle, break, burn or freeze your toe, the
perception is sensed by TRPV1 family-members, signaling to relevant
brain centers, which then generate tickle or pain perception to
your awareness. The toe will, however, swell because of local
release of small "neuropeptides" such as sP. They signal tissue
injury and trigger avoidance responses that favor the toe from
aggravation and repeat injury/stress.
[0030] One group of researchers that has conducted research on
diabetes for several decades studied insulin-producing beta cells
in the pancreatic islets of Langerhans almost ten years ago. These
researchers were puzzled by the observation of dense nervous system
elements in- and outside these islets (Winer et al., 2003.sup.20),
which float in a densely packed mass of exocrine pancreas tissue
that provides much of the digestive enzymes. Prominent in part of
this neuronal network were abundant TRPV1+ sensory nerves whose
roles were counterintuitive: what would TRPV1 heat sensors do in
the pancreas--a pancreas temperature of 45.degree. C. is not really
compatible with life.
[0031] In fact, it has been discovered (Razavi et al., 2006.sup.9),
that in mouse models, animals develop diabetes through progressive
loss of functional beta cells because their TRPV1 gene has two
coding mutations generating a secretory defect, releasing only
<5% of neuropeptides, including sP (Razavi et al., 2006.sup.9;
Tsui et al., 2008.sup.12; Tsui et al., 2007.sup.15): sP is a growth
and survival factor for beta cells. There is a local control
circuit, where local insulin ligates insulin receptors in TRPV1
terminals, which activates the channel to release local sP without
afferent signaling to the brain. In T1D-prone mice (and also in
diabetes patients, see below), beta cells for some time overcome
the sP secretory defect, inducing more sP release from "their"
TRPV1+ nerve endings with insulin. But too much insulin (called
hyperinsulinism) causes hypoglycemia, low blood sugar, and the rest
of the body fights that with growing resistance to insulin--a
progressively worsening cycle that stresses beta cells already
starved for their survival factor, sP. Hyperinsulinism and insulin
resistance well before disease onset are typical for the mouse
models and also for young patients with high type 1 diabetes risk
(Tsui et al., 2011.sup.14), in fact, it has been found that this is
true even for adult patients with Type 2 diabetes risk (Winer et
al., 2011.sup.18; Winer et al., 2009.sup.19).
[0032] Pancreatic delivery of substance P (sP) reverses recent
onset Type 1 diabetes mellitus (T1D) within hours in mice (Razavi
et al., Cell: 2006.sup.9). Near synchronous sP receptor binding in
pancreatic islets resets the regulatory circuit and globally
reverses beta cell stress. This presently proposed, initial,
translational study in T1D patients, aims to determine whether an
intra-arterial (i.a.) pancreatic sP injection is safe and carries
lasting promise for re-differentiation of beta cells and -function,
enhanced beta cell mass, reversing hyperinsulinism with
normalization of insulin resistance and glucose tolerance as well
as down-regulation of local diabetic autoimmunity, as it does in
relevant animal models (Tsui et al., Diabetes Metab. Res. Rev:
2011.sup.14).
[0033] Discovered about 80 years ago, substance P has been
extensively used in human clinical studies with at best rare
adverse events, in part reflecting its extremely short tissue
half-life of a minute or less. Decades of experience with human
clinical sP trials demonstrate its lack of toxicity. Publications
of a selection of more recent clinical sP trials, as well as
analytic texts on neuronal elements of T1D, of the remission
("honeymoon") phase of T1D and an overview of research into the
role of sP in pancreatitis are found in the Investigator's
Brochure, module 1.2.3 of the CTA proposal to Health Canada,
Clinical Trials Group.
[0034] sP is a non-toxic 11mer neuropeptide with extremely short
tissue half-life, released primarily by sensory afferent sensory
nerves. www.trials.gov still lists nearly 2000 current trials that
involve sP, sP-agonists and/or -antagonists, remarkably without
sP-attributable adverse events noted there or in the older
literature. Although reminiscent in this respect of a lack of
toxicity with other neuropeptides in clinical use (e.g. LHRH, TRH,
CRH, GHRH, somatostatin), sP's short tissue half-life has so far
precluded clinical utility. In the studies proposed here, that
short half-life becomes an advantage, permitting strictly local,
pancreatic drug delivery with little systemic spillage yet
excellent local efficacy (Razavi et al., Cell: 2006.sup.9; Tsui et
al., Diabetes: 2008.sup.12; Tsui et al., Vanilloid Receptor TRPV1
in Drug Discovery: Targeting Pain and Other Pathological Disorders:
2010.sup.13; Tsui et al., Diabetes Metab Res Rev: 2011.sup.14; Tsui
et al., Ann. N. Y. Acad. Sci.: 2008.sup.16).
[0035] These animal data demonstrate that a single sP delivery to
the diabetic pancreas clears inflammatory lesions rapidly
(overnight) and lastingly, through two synergistic mechanisms: 1.
immediate relief of beta cell stress with lasting survival support
(Razavi et al., Cell: 2006.sup.9) and (likely) re-activation of
de-differentiated or quiescent beta cells (Talchai et al., Nat.
Genet.: 2012.sup.10; Talchai et al., Cell: 2012.sup.11); 2. rapid,
sP-receptor-mediated, selective apoptotic elimination of recently
activated, inflammatory lesions in pancreas and local lymphatic
tissue. I.a. pancreas injection of sP causes, like in all tissues,
brief (minutes), dose-dependent hyperemia. Under experimental
conditions, this can be expanded to a transient, pancreatitis-like
reaction by preceding injections of caerulein, a potent pancreas
toxin (Koh et al., J. Cell. Mol. Med.: 2011.sup.7). Without such
pretreatments, sP pancreas toxicity in rodents has not been
observed and it has been found that even large sP doses were not
pancreas toxic in a large animal (dog) study. To extend knowledge
of potential toxicity to children is a major goal of the present
study.
[0036] Recently, this has, for essentially the first time, been
achieved in animal models, providing a strong rationale and impetus
to translate this physiological, therapeutic strategy to patients,
without systemic toxicities, based on thousands of previous sP
trials.
[0037] In one embodiment of the present invention, as shown in FIG.
1, a catheter 112 containing a pharmaceutical composition is used
to inject a therapeutically effective amount of the pharmaceutical
composition into the celiac artery 114 of an individual to thereby
partially reverse recent onset Type 1 Diabetes (T1D). Catheter 112
is attached to a supply (not shown in FIG. 1) of the pharmaceutical
composition Also shown in FIG. 1 is the pancreas 122, spleen 124,
stomach 126, duodenum 128, lienal artery 142, portal vein 144 and
lienal vein 146.
[0038] In one embodiment of the present invention, the
pharmaceutical composition so injected may completely reverse
recent onset T1D. In one embodiment of the present invention, the
composition may transiently for months to years reverses recent
onset T1D. In one embodiment of the present invention, the
pharmaceutical composition aids storage, production and release of
insulin by beta cells in a pancreas. In one embodiment of the
present invention, the pharmaceutical composition comprises a
neuropeptide. In one embodiment of the present invention, the
pharmaceutical composition comprises substance P. In one embodiment
of the present invention, substance P is dissolved in saline in the
pharmaceutical composition.
[0039] In one embodiment of the present invention, the individual
treated as shown in FIG. 1 is diagnosed with Type 1 Diabetes (T1D).
In one embodiment of the present invention the individual is a
human. In one embodiment, when the individual is a human,
therapeutically effective amount of the pharmaceutical composition
is at least 10 nM/kg. In one embodiment, when the individual is a
human, therapeutically effective amount of the pharmaceutical
composition is at least 50 nM/kg. In one embodiment, when the
individual is a human, therapeutically effective amount of the
pharmaceutical composition is at least 100 nM/kg. In one
embodiment, when the individual is a human, therapeutically
effective amount of the pharmaceutical composition is at least 250
nM/kg.
[0040] Unexpected neuronal elements in diabetes development have
been discovered (Tsui et al., Rev Endocr. Metab. Disord.: 2003;
Winer et al., Nat Med: 2003.sup.17. Subsequently, it has been
demonstrated (Razavi et al., Cell: 2006.sup.9) that a critical
element of T1D development is pancreatic sP deficiency, reflective
of mutations in the NOD mouse TRPV1 gene. These mutations generate
a hypofunctional/hyposecretory phenotype (Tsui et al., Vanilloid
Receptor TRPV1 in Drug Discovery: Targeting Pain and Other
Pathological Disorders: 2010.sup.13; Tsui et al., Trends Mol. Med.:
2007.sup.15).
[0041] A single, pancreatic sP injection via the celiac artery
reversed recent onset T1D in NOD mice for 4-6 months without any
insulin therapy, a period approximately equivalent to about 6-8
years in humans (Tsui et al., Diabetes: 2008.sup.12).
[0042] Human TRPV1 is extremely polymorphic, with thousands of
different allele combinations in Caucasian and Asian populations,
while it is a monomorphic gene in regions of low T1D incidence,
e.g. Africa (Dorfman et al., Vanilloid Receptor TRPV1 in Drug
Discovery: Targeting Pain and Other Pathological Disorders:
2010.sup.4). Sequencing of over 8000 Caucasian T1D patients and
controls demonstrates significant bias in TRPV1 allele selection,
which share predominantly hypofunctional phenotypes in transfection
studies (unpublished interim results, Toronto-Pittsburgh-Helsinki
TRPV1 consortium).
[0043] It has been directly demonstrated that systemic sP
deficiency by a newly developed HNK1R (sP receptor) ligation assay
in the majority of T1D patients, finding intermediate levels in a
sizable subset of first degree relatives (Cheung, R. K. et al.,
manuscript in preparation).
[0044] The fundamental link between TRPV1, beta cells and
autoimmunity previously discovered extends to metabolic syndrome
and Type 2 diabetes, emphasizing the fundamental role of neuronal
controls in diabetes endocrinology (Tsui et al., Diabetes Metab Res
Rev: 2011.sup.14; Winer et al., Nat Med: 2011.sup.18; Winer et al.,
Nat Med: 2009.sup.19).
[0045] A large animal study in dogs has recently been completed,
seeking evidence for sP-induced pancreas toxicity following
pancreatic injection of sP via the celiac artery. No drug-induced
toxicity was found over a study-relevant dose range, using a broad
screen of repetitive biochemical blood tests (>1200 assays) as
well as extensive histopathology studies conducted blindly at the
University of Toronto Center for Phenogenomics by veterinary
pathologists NOT associated with the proposed trial. The injections
were conducted by the Sick Kids' clinical imaging team. Since this
VanilloidGenetics-sponsored study is unlikely to be published by
itself, excerpt imaging data are provided in the Protocol Addendum,
showing the pancreas-selective, rapidly transient (.about.7 min) sP
effect as measured with a co-injected, inert blue dye marker. The
short-lived, physiological sP responses
(hyperemia.fwdarw.pancreas.fwdarw.selective hyperperfusion and
extravasation.fwdarw.normal pancreas appearance) illustrate the
core effects expected to be duplicated in T1D patients.
[0046] The study has 3 stages (A, B, C), applying a cross-over
design where all patients ultimately receive the intervention:
Stage A, toxicity and sP dose finding, 12 patients; Stage B,
limited efficacy test, 40 randomized patients, cross-over design;
Stage C, follow-up to 6 months, all patients, possible extension of
follow-up by 6 months.
[0047] The primary objective of Stage A is to determine if there
are unexpected adverse events with pancreatic delivery of sP in
recent (<30 months) onset Type 1 Diabetic patients with basal
c-Peptide levels of .gtoreq.0.2 pmoles/mL. The second objective of
Stage A is to determine whether sP injection at doses of 10, 50,
100 or 250 nmoles/kg BW (3 patients per dose) improve insulin need
and/or basal or stimulated c-Peptide levels comparing pre-injection
and day 20-22 post-injection mixed meal tolerance (MMTT) data. If
one or more Stage A patients initially received an ineffectively
small dose, an effective dose may be offered towards the end of the
trial.
[0048] The primary objective of Stage B is to determine the
prevalence of sP-treated patients whose stimulated (MMTT) c-Peptide
levels are significantly elevated above base line, 3 weeks
post-intervention ("Responders"). The secondary objective of Stage
B is to determine if Responders show reduced insulin need, improved
hyperinsulinism and improved/normalized glycemia, 3 weeks
post-intervention (MMTT data).
[0049] The primary objective of Stage C is to determine if
Responders sustain treatment effects at 6 months post intervention,
or longer, if follow up is extended with a pre-planned
DSMB-approved protocol change.
[0050] These study objectives acknowledge the realities of an
initial translational effort with a stepwise approach to test its
potential clinical utility, confirm the absence of unexpected
toxicities and generate tentative answers to two core functional
questions: does human T1D respond to sP like mouse T1D, and what is
the likely longevity of such effects?
[0051] The study objectives parallel previously published animal
studies as closely as possible (Razavi et al., Cell: 2006.sup.9;
Tsui et al., Vanilloid Receptor TRPV1 in Drug Discovery: Targeting
Pain and Other Pathological Disorders: 2010.sup.13; Tsui et al.,
Trends Mol. Med.: 2007.sup.15; Tsui et al., Ann N Y Acad. Sci.:
2008.sup.16). Clinically, this post-onset study is based on 2012
publications--an NIH-TrialNet meta-study of the first 2 years after
T1D onset (Greenbaum et al., Diabetes: 2012.sup.5) and research
reports collectively documenting the unexpectedly slow beta cell
death/loss post onset (Talchai et al., Nat. Genet.: 2012.sup.10;
Talchai et al., Cell: 2012.sup.11).
[0052] One of the several ways to confirm various conclusions about
sP (Tsui, 2010.sup.13; Tsui et al., 2008.sup.13) was to inject sP
into the pancreas of newly diabetic mice, a rather benign therapy
as sP is a physiological, non-toxic molecule that is quite fragile
and inactivated in tissue by dedicated and non-specific proteolytic
degradation as well as by binding to common NK1R in tissue within
seconds. As shown in FIG. 2, sP-injected diabetic mice rapidly
responded to the treatment, normalizing blood sugars and other
metabolic abnormalities typical for acute diabetes. Control animals
injected with vehicle (saline) only rapidly succumbed from
diabetes. In some way reminiscent of re-booting a crashed computer,
sP injection had lasting effects re-establishing normal insulin
control for months, although diabetes would eventually recur--but
will again respond to a single sP injection.
[0053] In this, the action of sP has several aspects, prominently
acting still as beta cell survival factor promoting beta cell
regeneration/re-differentiation. The unexpected bonus was, however,
that hemopoietic cells, including autoreactive lymphocytes that
target islets and beta cells, are for a brief time during
activation highly sensitive to sP: binding to sP receptors for a
brief period sets a death signal that kills the respective
lymphocyte via a form of apoptotic, programmed death response. In
fact, the immune system normally employs sP to terminate every-day
immune responses: in inflammatory tissue undergoing an immune
response, it is necessary not only to initiate but to limit that
process. FIG. 3 is a snap shot of what happens in pancreatic lymph
nodes, the "breeding place" for autoimmune lymphocytes. The in vivo
proliferation of these autoimmune T cells were measured as waves of
clonal expansion by cell division, each of the peaks in the figure
represents 1 cell division inside the pancreatic lymph node: there
were many divisions in the diabetic controls (312), i.e. they
rapidly generate many more autoimmune T cells that can kill
insulin-secreting beta cells. As shown in FIG. 3, most T cells in
sP treated mice (314) did not divide at all (pink shading) in fact
they die. While sP-treated mice that also received an sP receptor
(NK1R) antagonist (316) showed survival of at least two thirds of
the pathogenic T cell clones. Thus, sP injections promote beta cell
survival, regeneration and re-differentiation, while, at the same
time, selectively eliminating autoimmune cell pools that mediate
beta cell death and diabetes (Razavi et al., 2006.sup.9; Tsui et
al., 2008.sup.12; Tsui et al., 2007.sup.15).
[0054] In previous studies of intra-arterial (celiac artery) sP
injection into recent-onset diabetic mice (Razavi et al.,
2006.sup.9), pancreas pathology consistent with pancreatitis over a
dose range of 0.01-250 nM sP/kg has not been observed (the standard
dose was 1-10 nM/kg).
[0055] FIG. 4 shows islets from mice with recent onset Type 1
diabetes. The top shows a clean, no longer inflamed islet of an sP
injected mouse (1 nM sP/kg). FIG. 5 shows an infiltrated islet from
a control, diabetic mouse injected with vehicle (saline). Both
islets are surrounded by normal exocrine pancreas tissue without
any lesions typical for pancreatitis.
[0056] A large animal study of pancreatic sP injection in dogs,
using modern pancreatitis diagnostics is currently being conducted
(Mansfield et al., 2011.sup.33) and histopathology for the
detection of possible pancreatitis lesions. Data are expected to be
complete within about two weeks of this writing, in time for the
anticipated Pre-CTA meeting. Dogs are a rational choice of large
animal experimental model, since Dogs spontaneously develop
pancreatitis, have successfully been used in studies of
experimental pancreatitis and well tested diagnostics are available
(Trivedi et al., 2011.sup.34).
[0057] The typical, mostly rodent, studies of pancreatitis
induction that focus on the role of sP, demonstrated the
vasodilation effect of the drug and, in fact, previously published
sP injection data documented the same, using the commonly employed
Evans Blue dye tracing strategy (Razavi et al., 2006.sup.9), see
FIG. 5. In FIG. 5, pancreatic tissue is indicated by arrow 512.
[0058] Beyond brief, transient (minutes) tissue hyperperfusion
following sP injection, the drug does not cause tissue damage or
pancreatitis-like disease. For disease induction, the standard
experimental pancreatitis model employs multiple high dose
injections of the toxic secretagogue caerulein (Koh et al.,
2011.sup.7).
[0059] This protocol duplicates many aspects of spontaneous
pancreatitis, including elevation of both, endogenous sP and sP
receptor expression in pancreatic acinar cells, the active site of
pancreatitis development. In the exocrine pancreas, elevation of
sP/sP receptor is part of the NF.kappa.B pro-inflammatory
transcription pathway and begins to play a role in disease
progression 3-4 days after induction with caerulein (Chan and
Leung, 2011.sup.31; Hasel et al., 2005.sup.36; Wan et al.,
2008.sup.35).
[0060] While sP is one of the many elements that characterize the
inflammatory pancreatitis lesion, it does not elicit activation of
this transcription pathway in acinar cells. While sP has long been
a member of the multi-molecular response program caused by tissue
injury, binding of sP to its receptor, NK1R, in fact can transmit a
potent anti-inflammatory net signal that explains the enhanced
severity of some inflammatory responses to tissue damage in
NK1R.sup.null knock-out mice (Dib et al., 2009.sup.32).
[0061] The injection protocol is described as: Following placing
the femoral catheter to the celiac artery under real-time imaging
control, the dose finding sP amounts or the Stage B study
intervention dose (10, 50, 100 or 250 nM sP/kg BW, dissolved in 5
or 10 mL saline (see above), are injected over a 3 minute period.
The rationale: Steady rate injection was used in all mouse studies,
alternative rate injection timing were compared in the canine study
without finding any differences.
[0062] In one embodiment of the present invention, the amount of sP
administered to a patient may be 10-100 nM sP/kg BW, dissolved in 5
or 10 ml saline.
[0063] In another embodiment of the present invention, the amount
of sP administered to a patient may be 100-250 nM sP/kg BW,
dissolved in 5 or 10 ml saline.
[0064] As shown in FIG. 6, the overall study plan is based on the
assumption that if the intervention has effects comparable to those
observed in NOD mice, these should at least to some extent resemble
that biology. Thus, the desired effects on T1D endocrinology
should: (1) occur rapidly after injection of the short lived sP
(612) and (2) c-Peptide (or the other measures (see legend of FIG.
6) should follow one or more possible outcomes shown (622, 624,
626, and 628).
[0065] These outcomes are measured by a mixed meal tolerance test,
MMTT, on day 20 post-intervention (632, 634, 636, and 638).
Responders are defined to follow lines A, B or C, non-responders,
line D. All patients are then be entered into the 6 month follow
up, Stage C, under standard care--although surveillance and therapy
of Responders might have to be modified, as dictated by their T1D
status.
[0066] This study is described as open-label, randomized, crossover
clinical trial. Minimal Duration: 6 month after last patient
received intervention; extension of Stage C: Follow-up to 12 months
should be considered if treatment responders are identified.
[0067] Stage A is a dose finding and toxicity study. This stage
determines if a single sP dose of 10, 50, 100 or 250 nM sP/kg BW
(1) generates biochemical signs of pancreatitis or other organ
dysfunctions in eligible trial patients; or (2) improves
endocrine/metabolic T1D parameters. These doses were well tolerated
in rodent and canine studies (Module 1.2.3.5). Each of these
patients undergoes an MMTT 7-14 d before receiving the
intervention. The dose range was selected from animal experience:
50-250 nmoles/kg were equally effective and lasting, 10 less and 1
nmoles marginally so. Four patient groups of three are be injected
with clinical trial-grade sP, in each dose group. VanilloidGenetics
Inc has designed and outsourced the manufacture and validation of a
clinical grade injectable, pediatric (sP-ped) and a
regular/adolescent injectable drug formulation (sP-R/A) in a Health
Canada-approved, commercial cGMP facility.
[0068] Stage A patients are recruited first and randomized when
consented to a given sP dose (SASS procedure)--there is not a
placebo. Starting with the lowest dose, three patients receive 10
nmoles sP/kg, the next three, 50 nmoles sP/kg, followed by 100 and
250 nmoles sP/kg each, in the last two groups.
[0069] Stage B patients are randomized to the 1st or 2nd treatment
group. Based on animal data, it is expected that the maximal
response to be observed at 50-100 nmoles/kg. Study physicians,
together with the IGT angiographic team determines on clinical
grounds if patients receive the intervention in 5 or 10 mL saline
solution. At least 2 to 3 days of observation between groups is
planned, but this period may be modified by study management as the
clinical experience grows. Blood chemistries relevant to pancreas
and liver and enteric function are monitored after injection.
Patients are monitored (see below, standard care) for insulin need
and euglycemia.
[0070] The rationale behind Stage A is that Stage A generates the
first patient data. T1D reversal in recent onset diabetic mice
occurs within hours along mechanistic pathways quite well
understood. It is expected that sP effects might be rather prompt
in patients as well, given the fast kinetics of sP-receptor
ligation with its positive survival effects in beta cells and its
negative survival effects on the pancreatic autoimmune-infiltrate.
The Stage A strategy covers a judicious drug dose range and it
proceeds cautiously--although there is no reason to expect any drug
toxicities--and it generates the experience needed and the drug
dose used for Stage B. The Stage A strategy and cohort size was
discussed and incorporated in the final minutes of a pre-CTA
meeting with Health Canada (Investigators Brochure, Module
1.2.3.1). In this pre-CTA meeting, it was also discussed that if an
effective sP dose emerges in Stage A, it would be ethical to offer
Stage A patients that received a lower dose, a second injection at
the optimal dose by the end of Stage B may be offered, about 4
weeks after study start and as scheduling allows. This does not
jeopardize conclusions derived from this study stage or the study
overall, as repeat injections after treatment failure are
ultimately part of the follow-up plan, albeit at intervals
anticipated in the range of years.
[0071] Stage B of the study is the intervention stage. Altogether
40 eligible patients with recent onset T1D receive the Stage A
derived sP dose of choice by cannulation of the celiac artery under
imaging guidance. The rationale: T1D reversal is an essentially
binary (yes/no) initial response to sP therapy. Human T1D onset
differs from diabetes-prone mice by the frequent (.about.70%)
occurrence of a transient remission phase ("honeymoon", see below)
that begins sometime in the 1st year post onset and lasts from
weeks to (sometimes) months (Module 1.2.3.3). In the present study,
disease remission (euglycemia off insulin) might be difficult to
distinguish from spontaneous honeymoon, except that it is expected
to manifest in close proximity post-injection. While this phase-1
study cannot be aimed to generate significant efficacy data, the
control cohort(s) make it possible to elucidate trends in a
definitive fashion, in particular if patient treatment responses
are also binary and if sP-induced remission has anywhere near the
longevity of the murine therapy (equivalent to 6-8 human years),
more common relapse and re-injection after even 1 year may
clinically still represent a desirable effect, in particular if
non-invasive drug delivery strategies, now in development, can be
moved to application.
[0072] The next stage of the study, Stage C, is randomization. 20
eligible patients are recruited for randomization to intervention
and 20 as the control cohort in the initial intervention period,
terminated in each intervention patient 3 weeks later with an MMTT.
Thus analysis in the first intervention group has both,
pre-treatment and untreated control data.
[0073] About 1 week after the last intervention patient was
injected, the control cohort crosses over to intervention status,
undergo the pre-intervention MMTT, to receive the intervention
about a week later: this 2nd intervention period terminates with a
day 20 post-injection MMTT. Analysis of this group relies on
pre-treatment data as controls. The rationale: T1D reversal is an
essentially binary (yes/no) initial response to sP therapy.
[0074] Approximately 70% of patients with newly diagnosed Type 1
diabetes mellitus (T1D) temporarily restore to varying extent
endogenous beta cell function following the initiation of insulin
therapy (Bowden et al., 2008.sup.3). This period has been defined
clinically in several ways, but most commonly it is now called
"honeymoon" period, characterized by a daily insulin dose of
<0.5 units per kg body weight per day (U/kg/d) and
HbA1c.ltoreq.7.0% (Bowden et al., 2008.sup.3). During honeymoon,
blood glucose levels are frequently normal and stable--usually for
weeks, sometimes for months--with little or no need for exogenous
insulin and with near normal HbA1c, despite fluctuations in diet
and exercise. But by 6 months past onset, nearly all and by 12
months all but very rare patients will have reverted to
pre-honeymoon insulin needs and the full typical clinical T1D
course and signs (Abdul-Rasoul et al., 2006.sup.1).
[0075] The mechanisms governing this transiently improved beta-cell
function remain poorly understood. There is some consensus, that
hyperglycemia around disease onset represents added beta-cell
stress that functionally silences many beta cells still surviving
and that insulin therapy should relieve that stress to considerable
extent, sufficient for beta-cells to recover (or possibly even
regenerate/re-differentiate) (van Belle et al., 2011.sup.24).
Considerable normalization of elevated insulin resistance around
disease onset would also contribute to improved metabolic control
in honeymoon (Schober et al., 1984.sup.25), but it remains unclear
how the declaredly auto-aggressive immune system can temporarily
"shut down" its progressiveness: unfortunately there are few
relevant data sets and there is no animal model for honeymoon.
Nevertheless, the fact that honeymoon is common has generated
consensus, that it may represent the most promising target of
intervention therapies. Some 85% of new onset cases have no T1D
family history and onset is the first time diabetes declares
itself: pre-diabetes has no obvious symptoms for many years.
[0076] Unfortunately, as aptly implied by the term, honeymoon has
great variability in extent and duration, lasting anywhere from
weeks to months. Researchers have observed one exceedingly rare
case of honeymoon-like relapsing-remitting T1D that lasted well
over three decades before T1D was stable, with two of three
offspring from this patient developing full T1D early and without
honeymoon (Dosch et al. unpublished). Although extremely rare, such
cases illustrate that physiological mechanisms exist, able to curb
autoimmune progression, making successful intervention strategies
an ultimately attainable goal.
[0077] Because the honeymoon remission phase is a period of stable
metabolic control, it is important and promising to identify
factors that control the duration of clinical remission in T1D.
Several factors, including age, gender, pubertal status, metabolic
abnormalities at the time of onset, HLA genotype, presence of
diabetes-associated autoantibodies, have all been recognized to
affect the likelihood of partial or complete remission in newly
diabetic children (Buyukgebiz et al., 2001.sup.21). From the
discussion of published studies it becomes clear that there are
differences in age groupings and definitions that hinder firm
conclusions at this time.
[0078] The honeymoon phase often commences within days or weeks of
the start of insulin therapy, usually lasting for weeks, rarely for
months. In order to determine frequency and duration of the
honeymoon period after initiation of insulin therapy in newly
diagnosed patients, a group of 103 diabetic children, younger than
12 years of age, was prospectively monitored. Partial remission
occurred in 69%, complete remission occurred in three. The length
of time until remission was 28.6.+-.12 days, the honeymoon duration
was 7.2.+-.4.8 months (Abdul-Rasoul et al., 2006.sup.1).
[0079] A retrospective study involved 62 patients, diagnosed with
T1D under the age of 18 years during the years 1991-1998 (Bowden et
al., 2008.sup.3). Thirty-five patients (56.5%) entered partial
remission. The length of time until remission was 1.36.+-.1.03
months and positively correlated with insulin requirements at
discharge from hospital.
[0080] To determine whether there are different rates of partial
remission in preschool, school-age children and adolescents with
T1D, 152 consecutive patients with newly diagnosed T1D were studied
in 2004 (Bowden et al., 2008.sup.3). Patients were classified in
three age-groups (group-1 (<5 years), group-2 (5-12 years) and
group-3 (>12 years). Clinical characteristics at diagnosis,
hemoglobin A1C (HbA1C) and total daily insulin dose (TDD) were
analyzed in each age-class every three month over 1 year. Partial
remission was defined as TDD.ltoreq.0.5 units/kg/d, with
HbA1C<8%. Young children (group-1, 26.8%) and adolescents
(group-3, 29%) had low rates of partial remission compared with
school-age children (group-2, 56%, p=0.002). At 12 months, group
differences had disappeared: 13% (5/38) of group-1, 20% (11/56) of
group-2 and 18% (8/44) of group-3 remained in partial
remission.
[0081] The honeymoon period is different for each individual with
T1D, the variables that govern each course remain ill-defined and
different smaller studies can have considerably different outcomes.
Data sets were obtained from 6,123 pediatric T1D patients (<18
years old), who were treated in 157 pediatric centers and were
observed for 36 months at the same center starting from diagnosis
(Dost et al., 2007.sup.22). Analyses from this large multicenter
study of diabetic children included roles for age, gender and
pubertal status at onset of disease in shaping the amount of
insulin required and the clinical disease remission, which in this
study occurred during the first three years of the disease.
[0082] Multiple statistical analyses were performed to identify
factors influencing honeymoon duration. Results revealed that
partial remission (insulin<0.5 U/kg/d and HbA1c<7.0%)
developed in 1992 children (32.5%), most within the first 3 months
after diagnosis. Among those, 21% entering remission were younger
than 5 years, 37% were 5.sup.-10 years old, 37% of patients were
10-15 years old and 5% were adolescents, 15-17 years old at disease
onset. These analyses consolidate earlier report that the rates of
partial remission are higher in younger patients.
TABLE-US-00001 Age at onset <5 years 5-10 years <10 years
10-15 years >15 years Boys 10.4 .+-. 10.2 8.76 .+-. 9.24 9.36
.+-. 9.36 8.76 .+-. 9.24 11.04 .+-. 10.8 Girls 8.4 .+-. 9.2 7.56
.+-. 8.16 7.8 .+-. 8.52 9.36 .+-. 9.72 11.04 .+-. 10.08 P 0.013
0.085 0.0039 0.31 0.98
[0083] Remission lasted for an average of 0.74.+-.0.77 years (8.8
months) and was significantly shorter in children<10 years of
age at T1D onset, compared to patients with later onset, as shown
in the table above. The shorter honeymoon phase in younger children
might be related to a higher rate of ketoacidosis and/or a more
abnormal metabolic situation at diabetes onset. Children with
pubertal diabetes onset had a longer remission phase, conceivably
reflecting anti-inflammatory hormonal testosterone effects, since
there were gender differences in honeymoon duration, with longer
remission periods in boys (estrogen is more pro-inflammatory).
However, this gender effect was mainly observed in children<10
years old (p=0.0039), where sex hormone levels are low and no
significant gender difference was found in patients with diabetes
onset during or after puberty. Thus the role of hormonal effects in
onset and post-onset remission remains unclear.
[0084] The extent of metabolic imbalance at disease onset seems to
be an essential factor that determines the prevalence and length of
remission (Abdul-Rasoul et al., 2006.sup.1). The severity of
metabolic abnormalities at the time of diagnosis, i.e. the
relatively long duration of disease prior treatment, large blood
glucose elevations, high HbA1C values, the duration of glucosuria
and high insulin requirements to establish euglycemia (due to
insulin resistance) is associated with shorter clinical remission
in children with T1D (Vetter et al., 1982.sup.23).
[0085] Diabetic ketoacidosis (DKA) at diagnosis lowers the
honeymoon prevalence, perhaps reflective of a decreased capacity
for beta-cell recovery after the beginning of insulin therapy. DKA
is a consequence of insulin deficiency, corresponding hyperglycemia
with the burning of fatty acids accumulating systemic ketone
bodies, thus generating potentially fatal metabolic acidosis.
Children younger than 5 years or older than 12 years of age are
more likely to develop DKA than children between 5 and 12 years of
age, reasons are elusive (Bowden et al., 2008.sup.3)
[0086] Endogenous insulin secretion is assessed by measurement of
C-peptide. Coded by the insulin gene, the 31 amino acid C-peptide
is cleaved from pro-insulin and both are co-secreted at a
one-to-one molar ratio. Measurement of baseline and stimulated
C-peptide (after glucagon or a mixed meal) in patients with
recent-onset T1D is used as a measure of residual, global beta cell
function or -mass. Several studies have shown that higher C-peptide
levels are positively associated with honeymoon during the first
six months of T1D and C-peptide levels are a good predictor of
honeymoon during the first year of T1D (Zmyslowska et al.
2007.sup.26).
[0087] In a study of 268 patients with recently diagnosed T1D,
patients were stratified by gender, age, and season at diabetes
onset (Agner et al., 1987.sup.27). During the first 36 months of
disease, an assessment was performed for basal C-peptide, HbA1c,
and insulin dose per kilogram. Total disease remission was set as
complete discontinuation of insulin therapy for at least 1 week
with stable metabolic control, while partial remission was set as
an insulin need of .ltoreq.50% of the insulin dose at discharge
from the hospital. During the first 18 months of disease, 12.3% of
the patients developed total remission for a median of 6 months,
and 18.3% of patients developed partial remission, also for a
median of 6 months. Patients entering honeymoon had significantly
higher basal C-peptide levels than those who did not. From these,
unfortunately not too consonant studies, a general conclusion can
be drawn, that known factors predicting honeymoon and honeymoon
duration include pre-pubertal onset, male gender, mild initial
metabolic derangement and absence of frank ketoacidosis.
[0088] Overall, T1D honeymoon remains poorly understood, in
particular with respect to autoimmune progression and its almost
certain, but unproven, transient suppression. Nevertheless,
available data and broad consensus identify the honeymoon period a
natural and promising target of intervention therapies aimed at its
therapeutic extension. Islet autoimmunity does not disappear during
honeymoon, as judged by T1D-associated auto-antibodies, but clearly
lacks progression since there is sufficient endogenous beta-cell
derived insulin (and c-peptide) production. The honeymoon process
therefore must reflect the acute emergence of regulatory
lymphocytes which down-regulate autoimmune effector function--there
are no data, even cues why and how that would occur. The consistent
failures of toxic immunosuppression trials in recent-onset T1D
patients emphasize that overall conclusion: immunosuppression kills
regulatory lymphocyte pools as well as effector cells.
[0089] Collectively, the observation of honeymoon in a large
proportion of recent-onset T1D patients implies that physiological
mechanisms for breaking disease progression do exist and effective
interventions during this hold promise if they support the
physiological escape from disease progression obvious in honeymoon.
Such interventions must either slow or arrest the progression of
autoimmune beta-cell destruction/de-differentiation. The substance
P pancreas injection trial is the first to employ a non-toxic
physiological treatment strategy in T1D which is effective in
animal models to achieve just that target profile. It relieves the
chronic neuropeptide deficiency that characterizes the key genetic
T1D susceptibility in TRPV1-mutant rodents and hypofunctional TRPV1
allele selection in T1D-susceptible humans. This strategy promises
to precipitate and extend honeymoon by years and repeat injections
are effective in the same animal models. The hallmark T1D trials,
DCCT/EDIC, determined that even a limited (months) period of (near)
normal glucose metabolism has major impact on the devastating T1D
complications 2-4 decades later: while substance P therapy is not a
cure of the genetic underpinnings of T1D, it promises major,
positive, long-term impact on the harsh realities of living with
T1D and its enormous multi-billion annual costs long-term of
complications.
[0090] In one embodiment, the amount of the sP administered to an
individual is the range of 50-100 nM/kg.
EXAMPLES
Example 1
Study Cohorts--Sample Size N=52
[0091] 52 eligible, consented children or adolescents aged 10-18
years are recruited with informed, parental consent and patient
assent, as appropriate. These patients have been diagnosed with T1D
within .ltoreq.30 months and have a basal c-Peptide level at
recruitment of .gtoreq.0.2 pmoles/mL.
[0092] Patients with recent onset T1D (CDA guidelines
http://www.diabetes.ca/for-professionals/resources/2008-cpg/, see
below), with positive measures of typical metabolic dysfunction,
insulin replacement need; T1D-associated B- & T cell
autoimmunity is added, as this T1D element is one of the direct
therapy targets. The patients were diagnosed at or referred to the
Hospital For Sick Children, Toronto, ON, e.g. by one of the
associated SickKids Satellite Diabetes Centers in the greater
Toronto area. Patients are eligible for the trial, provided that:
1. they are between 10-18 years old; 2. patients are .ltoreq.30
months from diagnosis and have no other chronic illnesses other
than treated hypothyroidism--not uncommon in T1D; 3.
parents/caregivers and, when agreeable to them, patients have
received verbal explanations of the trial and have viewed the
Health Canada approved, educational video describing sP therapy in
diabetes. Explanations include familiarization with the potential
risks associated with a visceral angiogram, the injection of sP,
anesthesia and the (in humans) untested possibility of intervention
failure, prior to providing their informed consent, in writing, for
the study and the procedural angiogram.
[0093] sP treatment may not benefit patients that have normal sP
secretory activity, controlled by the TRPV1 genotype. To stratify
the cohort in this small initial study for the most likely
responders, and to reduce possible risks for unlikely responders,
patients are eligible for intervention if they carry at least one
of the polymorphic, T1D-associated TRPV1 alleles that are prevalent
in T1D patients/families. Since the large TRPV1 sequencing program
in Europe and North America may remain incomplete for years to
come, it is possible that sequences may be encountered that are as
yet unclassified. Therefore, a bioassay has been developed that
measures occupancy status of lymphocyte NK1R (the main sP receptor
which internalizes rapidly following sP-ligation). Most T1D
patients have abnormally elevated levels of unoccupied surface
NK1R, reflective of low steady state sP levels, and this assay can
be used to confirm intervention eligibility. These data are
described and discussed in the HC Investigator Brochure.
[0094] Based on sequence and functional data, it is expected that
as many as 20% of possible recruits may fail this last, dual
inclusion set which is designed to focus the study on the
theoretically most likely sP-responsive population. Larger
follow-up studies may have the power to distinguish patient
subpopulations and emphasize or de-emphasize these inclusion
criteria. The rationale: T1D reversal is an essentially binary
(yes/no) initial response to sP therapy. Human T1D onset differs
from diabetes-prone mice by the frequent (.about.70%) occurrence of
a transient remission phase ("honeymoon") that begins sometime in
the 1st year post onset and lasts from weeks to months (Module
1.2.3.3). In the present study, disease remission (euglycemia off
insulin) is expected to be induced that is difficult to distinguish
from spontaneous honeymoon, except that it is expected to manifest
rapidly within hours-days post injection, rather than over a period
of days. While this phase-1 study cannot be aimed to generate
significant efficacy data, the control cohort(s) makes it possible
to elucidate trends in a definitive fashion, in particular if
patient treatment responses are also binary and if sP-induced
remission has anywhere near the longevity of the murine therapy
(equivalent to .about.6 human years, more common relapse and
re-injection after even 1 year may clinically still represent a
desirable effect, in particular if the non-invasive drug delivery
strategies in development can be developed.
[0095] A number of permanent, acquired or congenital as well as
transient conditions preclude participation in this study: 1. In
patients with the common, transient remission ("honeymoon", <0.5
U insulin/kg), there is no acute measure of drug effects and sP
intervention treatment may be delayed until disease relapses, using
rising insulin needs to .gtoreq.1 U/kg as relapse measure. 2.
Patients have known co-morbidities, including ACE-inhibitor treated
hypertension as well as chromosomal abnormalities, impacting one or
more organ systems. Common childhood infectious diseases with
fever.gtoreq.38.degree. C. would lead to re-scheduling. 3.
Pregnancy. 4. Patients with a known radiographic contrast allergy.
5. Overweight (BMI 85-95th percentile) or obese (BMI>95th
percentile for age) patients with a BMI>90th percentile for age
are not be eligible for this initial study, as c-Peptide levels are
modified by elevated weight. 6. Parents plan to leave Toronto
imminently and follow-up cannot be assured.
[0096] Since the preclinical and animal data (Razavi et al.,
2006.sup.9) indicate that the treatment is most likely to benefit
patients that have a reduced function of TRPV1 channel. Therefore,
the potential candidate must be a carrier of at least one
T1D-associated allele in the TRPV1 gene: rs8065080 c.1753A>C/T/G
(p.Ile585Leu/p.Ile585Phe/p.Ile585Val) or; rs224534 c.1406C>T
(p.Thr469Ile) or; rs222749 c.271C>T (p.Pro91Ser) or; rs222747
c.945G>C (p.Met315Ile). Sequence listings of PCR primers for
amplification and sequencing of specific TRPV1 missense variants
are provided (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8). All
PCR products, 500-688 bp long, are amplified from genomic DNA
extracted from buccal swab samples. Purified PCR products are
re-sequenced, by the certified Genomics Quebec facility, from both
ends and the consensus genotype is determined. Patient genotypes
are communicated only to the lead physician who, with his advisors,
confirms the eligibility for the trial based on patient's
genotype.
[0097] Prior to treatment all consented patients provide a buccal
swab sample for DNA analyses using an Oragene.TM. DNA Collection
kit. The DNA is used to amplify and sequence specific exons of the
TRPV1 gene to identify a patient's genotypes, which will be used to
determine the eligibility for the study. Patients with wild type
TRPV1 genotypes are not eligible for this trial, as they are
unlikely to benefit from the treatment and their T1D disease might
have developed due to alternative pathogenic mechanisms. FIG. 7
shows a DNA collection kit 702 that may be used in one embodiment
of the present invention to collect DNA samples. DNA collection kit
702 includes swabs 712, a tube 714, a funnel 716 that screws onto
tube 714, a funnel lid 718 that is connected to funnel 716 by a
flexible strap 720, and a small cap 722 for tube 714.sub.[MG1].
Although a particular DNA collection kit is shown in FIG. 7, other
types of DNA collection kits may be used in various embodiments of
the present invention to collect DNA samples.
Example 2
Drug Formulation
[0098] Liquid, for Injection, substance P in 0.9% Sodium Chloride,
for Injection USP. Sp-R/A.TM.: 2 mg sP/mL and sP-Ped.TM.: 0.5 mg
sP/mL, vials: 1 mL each. cGMP-grade active pharmaceutical
ingredient, API (Polypeptide Inc, San Diego, Calif., 98% pure),
cGMP sterile drug manufacture outsourced to Dalton PharmaServices,
Toronto. Production procedures and validation are complete and
follow industry standards; manufacturing data.
Example 3
Dosage Regimen
[0099] Dose finding and toxicity study--Stage A, 12 patients
randomized to receiving 10-50-100 or 250 nmoles/kg i.a., 3 patients
per dose group. The treatment dose was originally calculated from a
count of cell surface-expressed sP receptors, NK1R, per mg mouse
pancreas tissue, obtained from serial sections and extrapolated to
the whole organ--not a procedure that can be duplicated in T1D
patients. The sP dose was calculated to give a 100-fold saturation
per surface receptor extrapolating to 2 nM/20 g mouse, adjusting
for in-tissue dwelling time and drug half life. This dose was
indeed effective. New onset mouse T1D has a massive lymphocytic
infiltration and many of the pancreatic surface NK1R receptors are
actually carried by these infiltrating cells, in fact, most of
these cells are legitimate and almost certainly prerequisite
targets of sP therapy, as it triggers rapid lymphocyte death in
recently activated lymphocytes (Module 1.2.3.6). This dramatically
reduces autoimmune effector cells in the pancreas and its
associated lymph node tissue. The new onset pancreas inflammatory
lesion of human T1D may be less dense, but data available are
insufficient for exact comparisons, generating such data is not
presently feasible. The standard sP dose in mouse experiments was 2
nM/mouse (i.e. 100 nm sP/kg BW) where a dose of 0.2 nM/mouse had
only partial effects. On this background, it is estimated that a
dose of 100 nM/kg BW may provide a similar degree of acute NK1R
ligation in situ as was achieved in sP therapy-responsive new onset
T1D mice.
Example 4
Washout Period
[0100] Based on agreements during the pre-CTA meeting (May, 2012)
no placebo is used, and ultimately, all patients receive an sP
injection. However, if warranted by significant diabetes reversal
rates, patients in the Stage A dose finding group, who did receive
an ineffectively low sP dose, may be offered a second injection
towards the end of the intervention period. With a vascular
half-life time of as low as <30 seconds, up to a minute in some
locales, the washout period for sP would be extremely short.)
Example 5
Pre-Study Screening and Baseline Evaluation
[0101] Standard patients care, SOP at the Hospital, includes
initial as well as repeated measurements of a number of metabolic
variables, but in addition, by 1-5 weeks prior sP intervention, at
least one measurement is required of T1D-relevant B and T cell
autoreactivities, as well as one MMTT. Added tests, such as the
sequencing of TRPV1 allelic sequence diversity and of NK1R (sP
receptor) occupancy rates was described and discussed in exclusion
criteria, above.
Example 6
Treatment/Assessment Visits
[0102] A detailed description of the intervention and
post-intervention process is provided. As described, it may be
overly cautious and represent a patient stress that may well
outweigh that of the intervention itself: the routine femoral
injection procedure is usually done on an outpatient base. By the
end of the Stage A toxicity and dose finding period, it is
determined if the observation period and intensive supervision can
be safely reduced with DSMB agreement.
[0103] Study-relevant baseline data are important. As a rule, new
onset data are available in the patient charts. The study requires
the following blood tests, drawn 1-5 weeks before recruitment and
randomization: HbA1c, T1D-associated B- and T-cell autoreactivity,
pancreas and liver function tests and, critically, an MMTT,
generating data on c-Peptide (basal, peak, AUC), fasting and
stimulated insulin & glucose levels). The study aims to begin
Stage A (dosing, toxicity) when 12 patients are or about to signing
informed consent. Recruitment for Stage B (Intervention) continues
from there.
[0104] Duplication of sampling with standard care sampling is
avoided through routinely close coordination by the trial physician
and the trial Monitor. Research Pharmacy is notified of patient
scheduling, including Name, HSC ID, body weight (kg) 1-3 weeks
before scheduled intervention.
[0105] The regular (adolescent) sP formulation, sP-R/A.TM. (BLUE
label), contains 2 mg sP/mL in saline, the pediatric formulation,
sP-Ped.TM. (YELLOW label), 0.5 mg sP/mL. Both formulations are
stored frozen at below -20.degree. C.
[0106] The research pharmacy receives from the study staff, patient
ID, body weight and scheduling to re-calculate and confirm or
correct the intervention dose and the appropriate number of blue or
yellow vials, communicating by email with the trial physician by
day minus-2. An Excel.RTM.-based automated calculator has been
developed and validated by VanilloidGenetics Inc and was provided
to the study team. A written prescription order, stating patient
Name, ID, BW and the final dose as well as the volumes from
blue-coded sP-R/A and/or yellow-coded sP-Ped to be combined in a 10
mL sterile syringe. On the morning of intervention, the required
drug vials are removed from its secure cold storage, signed out of
storage, placed in a sealed, sterile plastic bag, thawed at room
temperature and stored on water-ice until signed over to trial
staff, in this case the lead physician and the trial monitor. The
ready-to-use vials are transported to the intervention OR and
delivered to the IGT team performing the injection.
[0107] On the morning of study entry (day-1), consented T1D
patients have a scheduled fasting blood glucose measurement. In the
rare case where a recent MMTT dataset is not available, such a
patient undergoes an MMTT. After the MMTT, a modified insulin dose
may be prescribed if required and patients are released to go home,
after seeing an anesthesia and IGT consult to discuss the next day
intervention, unless these meetings have already been concluded
previously.
[0108] The next morning, fasted intervention patients are admitted
to hospital between 8-9 am. Provided that it has been confirmed
that the intervention drug is on-hand from the Pharmacy. Patients
are prepared for and receive brief, standard anesthesia for
delivery of sP via the celiac artery. The IGT imaging team has
routinely carried out femoral angiograms in their clinical practice
as well as performing sP celiac injections for the entire canine
study without adverse events. Stage A patients are randomized to
one of 4 groups, 3 patients each, to receive 10, 50, 100 or 250
nmoles sP/kg. The given dose is injected and patients enter a
monitoring phase, initially leaving 2d between groups, as permitted
by OR availability and scheduling constraints. The data generated
in Stage A, in particular improved fasting glucose levels and
reduction in insulin need are used to identify possible sP dose
responses. All daily glucometer readings are uploaded frequently to
the study site. The 3-4 initial (Stage A) patients are admitted
overnight and then stay near the Hospital for at least the first 2
days, as decided on clinical grounds by the PI. The conclusion of
Stage-A is expected to identify the single Stage B intervention
dose, a process that involves staff, and the study board. The
rationale: Stage A generates the first patient data. T1D reversal
in recent onset diabetic mice occurs within hours along mechanistic
pathways quite well understood. It is expected that sP effects
might be rather prompt in patients as well, given the fast kinetics
of sP-receptor ligation with its positive survival effects in beta
cells and its negative survival effects on the pancreatic
autoimmune-infiltrate. The Stage-A strategy covers a judicious drug
dose range and it proceeds cautiously although there is no reason
to expect any drug toxicities--and it generates the experience
needed and the drug dose used for Stage B. The stage A strategy and
cohort size was discussed, approved and incorporated in the final
minutes of the pre-CTA meeting with Health Canada (Investigators
Brochure, Module 1.2.3.1). In this pre-CTA meeting, it was also
discussed that if an effective sP dose emerges in Stage A, it would
be ethical to offer Stage A patients that received a lower dose, a
second injection at the optimal dose by the end of Stage B, about 4
weeks after study start and as scheduling allows. This does not
jeopardize conclusions derived from this study stage or the study
overall, as repeat injections after treatment failure are
ultimately part of the follow-up plan, albeit at intervals
anticipated in the range of years.
[0109] After completion of Stage A, these patients enter Stage C
(follow up), unless it is decided to offer those patients that
received a suboptimal dose a second injection with the Stage B
dose. Such patients might be added to an intervention cohort in
Stage B. During completion of injections in Stage A, recruitment
for Stage B continues: pre-set randomization is done in groups of 5
patients each for the intervention and control cohorts. Stage B
interventions begin when the first group of 10 (5 controls, 5
intervention) has been consented, and it continues until 40 gave
consent--unless unexpected adverse events trigger a study
moratorium or DSMB-sanctioned protocol changes. The DSMB has been
constituted with three senior, academically well respected T1D
physicians from the U.S and one from Europe: the DSMB will
communicate frequently with the study team and as a board via
Skype. All observations made in the Stage B toxicity and
dose-finding study, as well as any intervention-ascribed adverse
events are communicated promptly to the DSMB, advisory board
(email, phone) and adverse, in particular sP-associated events to
the REB: there now is an on-line reporting path for adverse events
to Health Canada. The DSMB has the power and obligation to stop or
modify the trial in face of unexpected adverse events. In the
absence of intervention-ascribed adverse events, 40 recruited and
eligible patients then receive the study dose in a modified
cross-over study design: 20 intervention patients are controlled by
20 at that point un-injected, control patients, which in turn cross
over to become intervention patients, controlled by their own
pre-intervention data, 3 weeks old.
[0110] The injection protocol is described as: following catheter
placement into the celiac artery, the dose finding sP amounts or
the Stage B study intervention dose (10, 50, 100 or 250 nM sP/kg
BW, dissolved in 5 or 10 mL saline (see above), are injected over a
3 minute period. The rationale: Steady rate injection was used in
all mouse studies, alternative rate injection timing were compared
in the canine study without finding any differences.
[0111] After introduction of patients and parents to the study, and
if interested in participating in the intervention and/or control
group, the parents and child are seen in the Hospital IGT Clinic,
in advance of the procedure, when a celiac angiogram is explained
in detail. The risks are outlined. These include local groin issues
(bleeding & hematoma, pseudoaneurysm, arterial dissection,
arterial thrombosis/stenosis, AV-fistula, pain); local issues in
the celiac territory (thrombosis, spasm, arterial dissection,
bleeding); systemic issues (contrast allergy, X-ray exposure, air
embolism, stroke); injection related (pancreatitis, liver
dysfunction, cholecystitis); innovative drug status (off label). An
informed consent is signed by the parents and if appropriate, the
patient assents.
[0112] Blood work is organized for the day prior to the procedure
including CBC, coagulation (INR, PTT) as well as typing and
screening for 0 units, these tests are in addition to those listed
above, but typing and screening can be done several weeks prior
intervention. The date of last menstrual period is checked for
girls>12 years and pregnancy tested in accordance with current
Hospital policy. A baseline diagnostic abdominal ultrasound is
arranged to specifically examine the pancreas, liver, gall bladder
and spleen. An anesthesia consult is organized prior to the
intervention.
Example 7
Concomitant Medication
[0113] Brief anesthesia during celiac angiogram and sP injection,
standard recovery, local pain management injection site, insulin
prescription based on frequent blood glucose measurements.
Example 8
Efficacy Variables and Analysis
[0114] The main measurements are MMTT-derived, diabetes-associated
variables prior to and after the 3 weeks post-intervention period,
the core variable for analysis being c-Peptide (basal, peak and
AUC). Secondary analysis includes insulin need, and estimates of
insulin resistance, fasting and stimulated blood glucose--unless
modified by clinical requirements, all values are derived from
MMTTs prior to, at the end of the intervention and at 6- and
possibly 12 months thereafter.
Example 9
Safety Variables and Analysis
[0115] Routine blood chemistries include two measurements of
pancreas, liver and intestinal markers during Stage A of the study.
If no abnormal values are observed, this number is reduced to one
measurement 4-7 d after intervention.
[0116] The study follows Health Canada ICH E2A reporting Standards.
Possible adverse events are monitored and recorded by trial staff
& Monitor. Two qualified physicians, NOT part of the study
team, form a stand-by AE team, one always on home-based call, their
call schedule posted to trial staff. Minor AEs are communicated for
discussion and analysis within 24 hr, at most 3 hr for
Serious/severe AE. All AEs are described, entered into study and
patient records, identified as study-related/unrelated, drug
treatment-related/unrelated using the SickKids adverse event form
(Study Protocol, pp.: 22). Serious/severe AEs are communicated to
the REB, HC and DSMB in writing.
Example 10
Statistical Analysis
[0117] This study implements recommendations of a recent
NIH-TrialNet T1D meta study, regarding clinical assay strategies,
statistical approaches and interpretations (Greenbaum et al.,
Diabetes: 2012.sup.5; Lachin et al., PLoS One: 2011.sup.8). For
Stage A (toxicity & dose finding), the first 12 patients
recruited are randomized (SAS procedure), each to one of the four
sP doses (10-50-100-250 nmoles/kg). All patients undergo a
pre-intervention MMTT prior intervention, the low dose first,
highest last, constituting the toxicity study. If during this dose
escalation process, there are drug-associated, unexpected adverse
events, the DSMB is consulted immediately to determine if further
dose escalation should continue. Stage A will be completed with an
MMTT 3 weeks after the last Stage A patient is injected, and before
the start of Stage B (intervention), although recruitment, consents
and pre-intervention MMTT will be obtained earlier. Stage B
(efficacy trends, n=40): In this two-sequence, two-period, single
treatment, modified crossover study, 40 patients are randomized
(SAS procedure) into the initial treatment or control groups (n=20
each). Patients in the treatment group undergo a baseline MMTT 7-30
days prior intervention.
[0118] These patients then receive the single-injection study
intervention with the sP dose selected in Stage A; 3 weeks later,
each treated patient undergoes an MMTT. This ends Stage B for the
given patient and provide all his/her data for the primary study
objective. These data are classifying that patient as responder or
non-responder based on c-Peptide values and lessened insulin need,
comparing the final MMTT to pre-intervention data as well as
relevant data sets from the control group. The treated patient then
crosses over to Stage C (follow up), with Home glucose measurements
and insulin doses are electronically uploaded regularly through a
secure study website and the data included in the analysis. The
frequency of glucose measurements may be determined by the PI, as
will be that of weekly-monthly phone contact between the study team
and the family (e.g. weekly, monthly).
[0119] The (so far untreated) control group undergoes an initial
MMTT during the second half of the initial intervention period I.
After completion of this period I, control patients cross over from
control to treatment status and be scheduled to receive the study
intervention (period II), which, for each patient, ends 3 weeks
after injection of the last patient, and completed with an MMTT:
pre- and post-intervention Data are compared as above, the patients
then crossing over to Stage C (follow up). Since each patient
receives only a single injection, the typical concerns about
"wash-out" periods between the 2 periods do not apply. In addition,
the intervention (sP) has an extremely short tissue half-life
(<1 minute). Formal data analysis is performed using the GEE
(generalized estimation equation) approach to build a
population-averaged marginal model. This model accounts for
correlation within the same patient and the time-varying covariate
(treatment effect). The primary measure is c-Peptide, basal or
stimulated, both measured in MMTTs and/or major reduction or
absence of insulin need. Secondary measures include insulin levels,
glycemia. Different correlation structures are investigated, and
the most appropriate correlation matrices are selected.
[0120] The final model estimates the treatment, period, sequence
effects, as well as relationships between treatment response, age,
age-at-onset, intervention time past onset, gender. The analysis
determines if any single or combined marker predicts sP
responsiveness/unresponsiveness. Hb1AC and autoimmune markers are
compared by repeated measures ANOVA pre-intervention and at 3 and 6
(and, if extended, 12) months of the study, as the systemic changes
measured are slower to emerge than immediate metabolic effects. If
there is consensus among the clinical team, advisory Board and
DSMB, the follow up period may be extended, using a monitoring
algorithm based on the study experience generated. The study should
terminate with an MMTT for final data analysis. SAS 9.3 and PROC
GEMOD with repeated statement are used throughout the analysis.
[0121] The statistical power of this small initial translation
study enables analysis in several ways. The scientific and clinical
need to monitor several key disease markers enhances the power in
combinatorial comparisons with Bonferroni corrections where
appropriate. At the 6-month study endpoint, with at best very few,
if any controls in honeymoon, three sP Responders would generate a
significant result. If sP therapy in fact generates a form of
honeymoon, where full remission is defined as <0.5 U
insulin/kg/d, approximately the same power would apply
proportionately. From previous experiences in the field (Herold et
al., N. Engl. J. Med.: 2002.sup.6), the analysis of secondary
outcomes, in particular HbA1c, c-Peptide (AUC, peak, fasting) may
be most sensitive and the several consecutive measurements in each
patient adds considerable power and confidence in the statistical
analysis despite its small size (Greenbaum et al., Diabetes:
2012.sup.5; Lachin et al., PLoS One: 2011.sup.8).
[0122] The brevity (3 wk) of the dosing and intervention stages, as
well as the non-intrusive character of the follow-up (Stage C,
uploading of glucometer and insulin data, 2 Hospital visits,
initially bi-weekly phone calls) is not likely to promote
loss-to-follow-up, (LTF). However, LTFs might be anticipated with
some preference in non-responders. If this scenario begins to
appear possible, despite extra efforts of the study team, and to
enhance the statistical power of the study, standard care Hospital
chart data from up to 30 T1D patients not related to the study but
followed by standard care at the Hospital since diagnosis over the
past 30 months are collected. Anonymized data sets on insulin use,
age at onset, fasting glucose and, where available, some oral
glucose tolerance test, are tabulated by a small team of students,
not otherwise related to the study.
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[0160] While the present invention has been disclosed with
references to certain embodiments, numerous modification,
alterations, and changes to the described embodiments are possible
without departing from the sphere and scope of the present
invention, as defined in the appended claims. Accordingly, it is
intended that the present invention not be limited to the described
embodiments, but that it has the full scope defined by the language
of the following claims, and equivalents thereof.
Sequence CWU 1
1
8124DNAArtificial SequenceDesigned PCR primer for amplification and
sequencing of specific TRPV1 missense variants. 1agaattgctt
gaacccagga ggca 24224DNAArtificial SequenceDesigned PCR primer for
amplification and sequencing of specific TRPV1 missense variants.
2agctgagaac cagcaaagca aacc 24324DNAArtificial SequenceDesigned PCR
primer for amplification and sequencing of specific TRPV1 missense
variants. 3tactttcaag cttgcctgcc ttgg 24424DNAArtificial
SequenceDesigned PCR primer for amplification and sequencing of
specific TRPV1 missense variants. 4attgtaagat gctccgcttg gcac
24524DNAArtificial SequenceDesigned PCR primer for amplification
and sequencing of specific TRPV1 missense variants. 5gcaaggatga
agaaatggag cagc 24624DNAArtificial SequenceDesigned PCR primer for
amplification and sequencing of specific TRPV1 missense variants.
6aagtccaagt gtctgtggct ggta 24724DNAArtificial SequenceDesigned PCR
primer for amplification and sequencing of specific TRPV1 missense
variants. 7tttgggcaga gacagaggga gttt 24824DNAArtificial
SequenceDesigned PCR primer for amplification and sequencing of
specific TRPV1 missense variants. 8ttcttctgtt ccaccctagg cagt
24
* * * * *
References