NZ616210B2 - Hepatocyte growth factor mimics as therapeutic agents - Google Patents
Hepatocyte growth factor mimics as therapeutic agents Download PDFInfo
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- NZ616210B2 NZ616210B2 NZ616210A NZ61621012A NZ616210B2 NZ 616210 B2 NZ616210 B2 NZ 616210B2 NZ 616210 A NZ616210 A NZ 616210A NZ 61621012 A NZ61621012 A NZ 61621012A NZ 616210 B2 NZ616210 B2 NZ 616210B2
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Abstract
Small molecule, peptidic hepatocyte growth factors mimics, which act as both mimetics and antagonists, have been generated and have the structure of formula (I) where R3 is isoluecine wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.. These molecules have been shown or predicted to have therapeutic potential for numerous pathologies including dementia, neurodegenerative disease, diabetes and metabolic syndrome, cancer, and defective wound healing. An example of a compound of formula (I) is N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide. These molecules have been shown or predicted to have therapeutic potential for numerous pathologies including dementia, neurodegenerative disease, diabetes and metabolic syndrome, cancer, and defective wound healing. An example of a compound of formula (I) is N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide.
Description
HEPATOCYTE GROWTH FACTOR MIMICS AS THERAPEUTIC AGENTS
STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made, in part, with government support under Grant Nos.
MH086032 awarded by NIH. The government has certain rights in the invention.
SEQUENCE LISTING
This application includes as the Sequence Listing the complete contents of the
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hereby incorporated by reference.
DESCRIPTION
SUMMARY
Field of the Invention
The invention generally relates to the development of hepatocyte growth factor (HGF)
mimics that can act as mimetics (agonists) or antagonists. Mimetics act: to enhance cognitive
function; as general neuroprotective/neuroregenerative agents; to facilitate wound repair; to
improve insulin sensitivity and glucose transport; and to decrease tissue or organ fibrosis in
order to prevent or reverse the symptoms of dementia, to protect from or reverse
neurodegenerative disease, to facilitate repair of traumatic injury to the nervous system, to
augment tissue and organ vascularization, to improve impaired wound healing, and to
decrease or reverse fibrotic changes in organs like heart, lung, kidney, and liver. Antagonists
act, for example, as anti-angiogenic and anti-cancer agents; to treat various malignancies and
diseases like macular degeneration and diabetic retinopathy, which are associated with
hypervascularization.
Mimetics:
Dementia: There are approximately 10 million diagnosed dementia patients in the United
States alone and that number continues to grow every year as the population ages. The costs
of treatment and care of these patients are in excess of $70 billion annually and are increasing
rapidly. Unfortunately, the current treatment options for the management of dementia are
severely limited and largely ineffective. The lack of treatment options for a burgeoning
health problem of this magnitude necessitates that new and innovative therapeutic approaches
be developed as quickly as possible.
At its core dementia results from a combination of diminished synaptic connectivity
among neurons and neuronal death in the entorhinal cortex, hippocampus and neocortex.
Therefore, an effective treatment would be expected to augment synaptic connectivity, protect
neurons from underlying death inducers, and stimulate the replacement of lost neurons from
preexisting pools of neural stem cells. These clinical endpoints advocate for the therapeutic
use of neurotrophic factors, which mediate neural development, neurogenesis,
neuroprotection, and synaptogenesis. Not unexpectedly neurotrophic factors have been
considered as treatment options for many neurodegenerative diseases including Alzheimer’s
disease (see reviews- Nagahara and Tuszynski, 2011; Calissano et al., 2010). One particularly
attractive but mostly overlooked neurotrophic factor is HGF, which has a proven ability to
both stimulate neurogenesis (Shang et al., 2011, Wang et al, 2011) and synaptogenesis (see
preliminary studies below). The realization that HGF application might represent a viable
treatment option for dementia should be no surprise. HGF is a potent neurotrophic factor in
many brain regions (Kato et al., 2009; Ebens et al., 1996), while affecting a variety of
neuronal cell types.
Neuroprotection/Neuroregeneration: HGF and c-Met are actively expressed in both the
developing and adult brains and nerves. The Met system is essential for both the central and
peripheral nervous systems to function properly. A large number of studies have shown that
HGF and c-Met are expressed in multiple areas of the brain including the frontal cortex,
subependyma, thalamus, cerebellar cortex, deep gray matter, and the hippocampus, an
important area for cognition.
The biological activities described above also characterize Met functions in the brain where
HGF/c-Met signaling is neurotrophic (Honda et al., 1995) and protective (Zhang et al., 2000;
Takeo et al., 2007; Tyndall and Walikonis, 2007; Takeuchi et al., 2008).Similar to its
activities in other tissues, Met in the brain is involved in development, acting as a guidance
factor during differentiation, motogenesis and neuritogenesis (Ebens et al., 1996; Sun et al.,
2002; Tyndall and Walikonis, 2007). HGF/ c-Met signaling has also been shown to promote
healing of neuronal injury (Trapp et al., 2008), especially after ischemic brain injury (Takeo
et al., 2007). HGF also displayed neuroprotective effects in animal models for
neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The various
functions of HGF, plus its highly potent neurotrophic activities, promote HGF as a potential
therapeutic agent for the treatment of various diseases of the nervous system.
Amyotrophic Lateral Sclerosis: ALS is a fatal rapid-onset neurodegenerative disease that is
characterized by degeneration of motoneurons in the spinal cord and efferent neurons in the
motor cortex and brainstem. The impact of this degeneration results in a progressive loss of
muscle function culminating in total paralysis. Approximately 90% of the cases of ALS are
classified as sporadic with no known etiology, while the remaining 10% appear to be familial,
resulting in part from defects in copper/zinc superoxide dismutase 1 (SOD1), which leads to
exaggerated oxidative stress and an unfolded protein response. The one thing that both forms
of ALS have in common is that there is currently is no effective treatment available.
Despite the paucity of effective treatment options, several studies have highlighted the
potential benefits of using hepatocyte growth factor (HGF) as a therapeutic agent. These
investigations have demonstrated that application of hepatocyte growth factor (HGF) in a
murine or rat model of familial ALS significantly slows motoneuron degeneration (Aoki et
al., 2009); reduces gliosis (Kadoyama et al. 2007), which contributes to the degeneration
process; delays the onset of paralysis (Kadayama et al., 2009); and increases lifespan (Sun et
al., 2002).
The realization that HGF application might represent a viable treatment option for
ALS, however, should be unexpected. HGF along with its type I tyrosine kinase receptor, c-
Met, have long been recognized for their role in the development of tubular structures (Santos
et al., 1993) and their general proliferative, anti-apoptotic, motogenic, and morphogenic
actions on hepatocytes and cells of epithelial origin . Most pertinent, however, is the more
recent realization that HGF is a potent neurotrophic factor (Maina and Klein, 1993; Kato et al.,
2009) in many brain regions and that it is particularly effective as a pro-survival/regenerative
factor for motoneurons (Ebens et al., 1996; Yamamoto et al., 1997; Hayashi et al., 2006; Elsen
et al., 2009).
Parkinson’s Disease: A treatment option long considered for many neurodegenerative diseases
including Parkinson’s disease (PD) has been the application of growth factors with the
intention of halting disease progression, restoring lost function, or hopefully both (review,
Rangasamy et al., 2010). However, this dream has gone largely unfulfilled at the level of
clinical medicine because of limitations related to brain delivery and costs. Growth factors are
universally large proteins that are both metabolically labile and too large to pass the blood-
brain barrier (BBB). As such, most approaches to delivery have utilized gene therapy methods
with the hope that the growth factor will be expressed in the correct location at a high enough
concentration and for a long enough period to provide clinical relief. Although a number of
creative and successful approaches in animal models have been employed to deliver growth
factors like GDNF (Wang et al., 2011) to the brain, these methodologies are technically
complex and prohibitively difficult to bring to practice with large numbers of patients.
While many growth factor systems have been examined as potential therapeutic targets
for PD one that has been largely, and we think mistakenly, overlooked is the hepatocyte
growth factor (HGF)/c-Met (its type I tyrosine kinase- receptor) system. Nevertheless, the
potential utility of HGF as a PD treatment has been highlighted in a study by Koike et al.
(2006) in which an HGF plasmid injected directly into the substantia nigra (SN) resulted in
localized over-expression of HGF, and acted dramatically to prevent neuronal cell death and
preserve normal motor function in the 6-hydroxydopamine (6-OHDA) PD rat model. This
observed neuroprotective effect of HGF on dopaminergic (DA) neurons meshes with its ability
to augment the proliferation and migration of dopaminergic progenitor cells (Lan et al., 2008)
The neuroprotective effect of the HGF on the nigrostriatal pathway, however, should
be no surprise given its recognized role in stem cell regulation, the development of tubular
structures (Santos et al., 1993) and its general proliferative, anti-apoptotic, motogenic, and
morphogenic actions on many cell types including hepatocytes and cells of epithelial origin
(Gherardi et al., 1993). Maina et al., Particularly pertinent is the demonstration that HGF is a
potent neurotrophic factor for many neuronal cell types (Kato et al, 2009) including
motoneurons ( Elsen et al., 2009; Hayashi et al, 2006), hippocampal neurons Lim et al., 2008),
cerebellar granular cells (Ieraci et al., 2002), and sympathetic neurons (1999). Moreover, HGF
appears to be a critical regulator of neural stem cell expansion and differentiation (Nicoleau et
al., 2009) suggesting that neural as well as many types of peripheral stem cells are under the
control of the HGF/c-Met system.
Traumatic Brain Injury/Spinal Cord Injury: TBI often negatively impacts cognitive function
and can elicit effects that range from mild, with temporary decrements in mental abilities, to
severe, with prolonged and debilitating cognitive dysfunction (Kane et al., 2011). Cognitive
difficulties along with other neurological deficits including: anxiety, aggressiveness, and
depression result in a significantly reduced quality of life (Masel and DeWitt, 2010). With
military operations concluded in Iraq and continuing in Afghanistan TBI has become the
major combat injury representing 28% of all combat casualties (Okie, 2005; U.S. Medicine,
May 2006, Vol 42). Total estimates of military service members suffering TBIs between
2001 and 2010 range from 180,000 to 320,000 (U.S. Defense and Veterans Brain Injury
Center).
Underlying TBI is physical injury to the brain resulting in decreased synaptic
connectivity among neurons, loss and death of neurons, damage to cerebral blood vessels
resulting in ischemic/hypoxic-induced damage, and secondary glial scaring. This loss of
neurons and diminished synaptic connectivity is particularly apparent in the hippocampus
(Gao et al., 2011; Zhang et al., 2011a; Zhang et al., 2011b) resulting in defective long-term
potentiation (Schwarzbach et al., 2006) and cognitive deficits (e.g. Dikmen et al., 2009; Patel
et al., 2010). The prevalence of TBI associated injuries that result in neuronal loss and
decreased synaptic connectivity denote the need for therapies which support neuronal repair
and/or replacement. These clinical endpoints advocate for the therapeutic use of neurotrophic
factors which mediate neural development, neurogenesis, neuroprotection, and
synaptogenesis, for treating TBI. Not unexpectedly neurotrophic factors have been considered
as treatment options for TBI (Kaplan et al., 2010; Richardson et al., 2010; Qi et al., 2011).
One particularly attractive but mostly overlooked neurotrophic factor is HGF, which has a
proven ability to both stimulate neurogenesis (Shang et al., 2011; Wang et al., 2011) and
synaptogenesis (see preliminary studies below). The fact that HGF application might
represent a viable treatment option for TBI stems from the recent realization that HGF is a
potent neurotrophic factor in many brain regions (Kato et al., 2009; Ebens et al, 1997), while
affecting a variety of neuronal cell types (Yamamoto et al., 1997; Hayashi et al., 2006; Elsen
et al., 2009).
HGF and wound healing: Excessive scarring is typified by unnecessary accumulation of
ECM components in the wound, due to an inappropriate balance between synthesis and
degradation. Therapy for pathologic scarring may be directed at inhibiting the synthesis and
promoting the degradation of the ECM. HGF in the skin promotes wound healing effectively
in several ways: motivating the proliferation and motility of dermal vascular endothelial cells;
stimulating the motility of epidermal keratinocytes; enhancing local blood supply; and
accelerating the re-epithelialization of the wound (Nakanishi et al., 2002). Re-
epithelialization inhibits the formation of scars. Studies have shown that HGF gene transfer
accelerates dermal wound healing by stimulating angiogenesis and reepithelialization
(Nakanishi et al., 2002). Therapeutic approaches that augment HGF/SF would be expected to
promote wound healing and prevent scar formation.
HGF as a treatment option for metabolic syndrome and diabetes: Several recent studies have
implicated the critical role of the HGF/c-Met system in the regulation of glucose handling,
insulin secretion, and tissue insulin sensitivity. Together these investigations have highlighted
the therapeutic potential of augmenting the HGF/c-Met system for the treatment of type 2
diabetes and metabolic syndrome (Fafalios et al., 2011; Flaquer et al., 2012)). These
investigators have shown that: 1) c-Met, the HGF receptor complexes with the insulin
receptor; 2) c-Met is critically involved with hepatic glucose homoestasis; 3) HGF restores
insulin responsiveness in a murine diabetic mouse model; 4) that HGF gene therapy can
prevent the renal damage that typically accompanies diabetes, and 5) HGF ameliorates the
vascular complication of diabetes (Peng et al., 2011).
The HGF/c-Met signaling pathway potentiating Angiogenesis: Angiogenesis is defined as the
formation of new blood vessels from existing vascular bed, It is a prime requirement in
physiological processes such as wound healing and the menstrual cycle, on the other hand, it
is an essential step for multiple pathological conditions, like cancer, macular degeneration,
atherosclerosis, diabetic retinopathy, neovascular glaucoma, psoriasis and rheumatoid
arthritis. Consequently, the modulation of angiogenesis, whether it was through encouraging
therapeutic angiogenesis or by stopping pathologic angiogenesis, is an exhilarating prospect
for modern medicine. The equilibrium between physiological and pathological angiogenesis
is mediated by the communication of numerous endogenous angiogenic and anti-angiogenic
modulators.
Numerous studies have shown HGF to be a powerful inducer of neovasculature
formation. Moreover HGF/c-Met inhibitors are clinically relevant anti-angiogenic agents.
(Gherardi et al, 2012).This is probably attained through multiple pathways, achieved either by
direct or indirect action on endothelial cells.
HGF as anti-fibrotic agent: Fibrotic disease takes many forms and is a major contributor to
degraded function in the heart, kidney, and liver secondary to many pathological states
including myocardial infarction, diabetes, and alcoholism. Hepatocyte growth factor (HGF) is
showing a strong anti-fibrotic effect with remarkable effectiveness in ameliorating tissue
fibrosis in a wide range of animal models HGF exhibits a remarkably powerful anti-fibrotic
effect that ameliorates tissue fibrosis in a wide range of animal models and tissues (Liu and
Yang, 2006). Evidence has documented the therapeutic effect of exogenous HGF in chronic
allograft nephropathic rats, a model of chronic inflammation and progressive tissue scarring.
The intramuscular administration of the human HGF gene reduced the rate of mortality,
restrained inflammation and infiltration, and reduced renal fibrosis (Liu and Yang, 2006).
Coronary artery disease (CAD) ischemic events and myocardial infarction are the
major causes of cardiac failure in the Western world. The only option for severe coronary
blockage and atherosclerosis is bypass surgery. Two pathological events in CAD play major
roles in the loss of cardiac function observed in CAD: 1) blockage of the coronary arteries
resulting in decreased blood perfusion to the heart; and 2) the formation of fibrotic tissue after
cardiac insult resulting in ventricle remodeling and decreased compliance. Increased levels of
HGF in the circulation have been reported after acute myocardial Infarction (Zhu et al., 2000;
Jin et al., 2003). This increase in circulating HGF can be used as biological marker for heart
injury and gives a clue regarding its protective role (Ueda et al., 2001). Pharmaceuticals that
enhance the HGF/Met signaling could potentially be used in the treatment of myocardial
infarction, providing protection against oxidative stress and cell death due to apoptosis as
well as reducing the formation of fibrotic tissue (Ahmet et al., 2002; Kondo et al., 2004;
Pietronave et al., 2010). Moreover, another beneficial effect of HGF following myocardial
infarction could lie in its ability to induce neovascularization, which could support formation
of new cardiac vasculature that would improve reperfusion of the myocardium.
Although HGF is known to protect the liver against external insults, HGF generation
has also been associated with several liver and extra-hepatic diseases. Experimental and
clinical evidence indicates that HGF plays a crucial role in liver regeneration. Liver cirrhosis
is the irreversible end result of fibrous scarring and hepatocellular regeneration and is a major
cause of morbidity and mortality worldwide with no effective therapy. Although there is no
specific etiology for this disease, cirrhosis has been defined as a chronic disease of the liver in
which dispersed damage and regeneration of hepatic parenchymal cells have taken place and
in which dissemination of connective tissue has resulted in inadequate organization of the
lobular and vascular structures (Fujimoto and Kaneda, 1999; Kaibori et al., 2002). Ideally,
approaches for the treatment of liver cirrhosis should include attenuation of fibrogenesis,
encouragement of hepatocyte mitosis, and reformation of tissue architecture.
Studies have shown that exogenous administration of recombinant HGF increases the
potential for liver regeneration after hepatoctomy especially in the cases of cirrhotic liver
(Boros and Miller, 1995; Kaibori et al., 2002; Borowiak et al., 2004). Conversely, studies
have shown that the clofibrate-related compounds, which increase HGF/SF levels, can induce
hepatomegaly, proliferation of hepatic peroxisomes, and hepatic carcinoma (Xu and Wu,
1999). The linkage of HGF/SF both positively and negatively to hepatic diseases has made
HGF-related therapeutics a hot area for pharmaceutical development.
Limitations to the direct use of HGF: The direct use of HGF or any other protein
neurotrophic factor as a therapeutic agent has two serious limitations: 1) large size and
hydrophilic character precluding blood-brain barrier permeability (BBB); and 2) the need to
be manufactured by recombinant methods at high cost, thus limiting its widespread use.
These impediments can be overcome using one or more of an extensive library of small
molecule HGF mimetics which are described herein, some of which are orally active,
display profound pro-cognitive/anti-dementia/ neuroprotective activity, and are
inexpensive to synthesize.
Antagonists: Improper activation of the c-Met receptor can be encouraged by genetic
activating mutations, transcriptional upregulation or by ligand-dependent autocrine or
paracrine mechanisms.
c-Met activation in cancer: Cancer is a heterogeneous group of diseases that result from the
accumulation of genetic mutations. These mutations cause altered function in proto-
oncogenes leading to dysregulation of DNA repair, proliferation, and apoptotic signaling
(Tannock, 2005). The dysregulation in the signals within a group of cells leads to the
uncontrolled growth, and invasion that either directly intrudes upon and destroys adjacent
tissue or metastasizes and spread to other location in the body through the lymphatic system
or the blood stream.
A dysfunctioning Met and HGF system appears to be a critical trait of numerous
human malignancies. Ectopical overexpression of HGF and/or c-Met in mouse and human
cell lines leads them to develop tumorigenic and metastatic phenotypes in athymic nude mice
(Rong et al., 1994). A large number of studies have shown that the HGF/c-Met pathway is
one of the most dysregulated pathways in human malignancies, which include, but are not
limited to: bladder, breast, cervical, colorectal, endometrial, esophageal, gastric, head and
neck, kidney, liver, lung, nasopharyngeal, ovarian, pancreatic, prostate, and thyroid cancers
(http://www.vai.org/met/). Lastly, an activating mutations of c-Met has been discovered in
sporadic and inherited forms of human renal papillary carcinomas (Danilkovitch-Miagkova
and Zbar, 2002). These mutations which alter sequences within the kinase domain have also
been found in other types of solid tumors and metastatic lesions. At this point it’s worth
mentioning that HGF over- or miss-expression often correlates with poor prognosis and that
the down-regulation of c-Met or HGF expression in human tumor cells reduced their
tumorigenicity (Abounader et al., 2002) .
Activation of Met in cancer occurs most often through ligand autocrine or paracrine
activation. Osteosarcomas and globlastoma mutliforme, which express both c-Met and HGF
are examples of dysfunctional autocrine control. In other instances where paracrine control is
paramount, c-Met over-expression has been reported in human primary tumors while HGF is
provided by stromal cells and not the tumor itself (Houldsworth et al., 1990; Kuniyasu et al.,
1992; Hara et al., 1998; Tong et al., 2004; Miller et al., 2006; Bean et al., 2007).
The list of neoplasms in which c-Met overexpression has been detected is growing
relentlessly. In the case of carcinomas, excessive levels of c-Met expression have been found
in virtually every malignancy (Danilkovitch-Miagkova and Zbar, 2002). Receptor over-
expression can lead to local receptor oligomerization generating cells reactive to sub-
threshold ligand concentrations. HGF itself is able to trigger the transcription of c-Met
(Boccaccio et al., 1994), and it is thus HGF, which is universally expressed by stromal cells
throughout the body that typically drives tumor over expression of c-Met (Aguirre Ghiso et
al., 1999; Parr et al., 2004). This uniqueness of HGF permits it to play a critical role, which
engages paracrine positive feedback loops that prop up the growth and metastasis of cancer
cells. Interestingly, this notion is in agreement with the observation that c-Met activating
mutations require HGF to enhance their catalytic effectiveness (Michieli et al., 1999).
HGF can also abnormally stimulate c-Met in an autocrine manner, as depicted in
gliobastomas (Weidner et al., 1990), breast carcinomas (Potempa and Ridley, 1998),
rhabdomyosarcomas (Hartmann et al., 1994) and osteosarcomas (Ridley et al., 1995). With
multiple mechanisms of activation, it is clear that both Met and HGF are major contributors
to the progression of most human cancers. Additionally, the demonstrated activities of c-Met
and HGF in proliferation, invasion, angiogenesis and anti-apoptosis (Weidner et al., 1990;
Rong et al., 1994; Kitamura et al., 2000; Xiao et al., 2001; Wang et al., 2002; Derksen et al.,
2003) demarcate the different stages at which these molecules can participate in tumor
development.
Although, c-Met is used as a general marker for cancer, is also an indicator of
biological significance with respect to malignancy and patient prognosis, with high levels
correlated with a poor prognosis. Molecules that inhibit c-Met and HGF can therefore be
expected to interfere with the molecular causes of many cancers, and should significantly help
in attenuating Recent studies from the Harding lab have confirmed the potential use of HGF
antagonists as effective anti-cancer/anti-angiogenic agents (Yamamoto et al., 2010, Kawas et
al., 2011; Kawas et al., 2012).
Macular degeneration/diabetic retinopathy: Age-related macular degeneration (ARMD) is
the most common cause of irreversible vision loss in Americans over the age of 60. It is
predicted that 10 million Americans will suffer from some level of this age-related visual
damage during their retirement years. In normal healthy eyes, retinal pigment epithelial (RPE)
cells form a polarized monolayer adjacent to the photoreceptors and are involved in various
activities that are essential to retinal homeostasis and visual function. In the case of macular
degeneration, unfortunately, adhesions and communication between RPE cells are lost
because of inflammation. When inflammation occurs, RPE cells secrete many growth factors
including HGF/SF, which stimulates the division and migration of RPE and the formation of
new vasculature from existing blood vessels (angiogenesis). HGF also stimulates the
production of other growth factors (e.g. VEGF), which further promote the formation of new
blood vessels that invade neighboring matrix (Jun et al., 2007). Hence the use of HGF
blockers could be used either prophylactically, or as a treatment to slow down the progression
of the disease and subsequent loss of vision.
Proliferative diabetic retinopathy (PDR), which entails a distinctive
neovascularization of the retina that is characterized by the invasion of vessels into the
vitreous cavity, is coupled with bleeding and scarring around the proliferative channel
(Katsura et al., 1998). There is substantial evidence that multiple growth factors are involved
in the onset and progression of the neovascularization process in general and in the PDR in
specifically. These include basic fibroblast growth factor (bFGF), Insulin-like growth factors
(IGF-I), vascular endothelial growth factor (VEGF), and HGF. Of these, HGF has the most
pronounced effects on endothelial growth and mitogenic activity (Boulton, 1999). Studies
have found that levels of HGF in the vitreous fluid of PDR patients are considerably higher
than in non-diabetic patients, and that the levels of HGF are especially high in the active stage
of PDR (Katsura et al., 1998). This suggests that HGF stimulates or perpetuates
neovascularization in PDR. Therefore, it is plausible to think that an HGF antagonist would
be a promising option as a prophylactic treatment, or to ameliorate the progression of PDR.
Accordingly, in a first aspect, the present invention provides a hepatocyte growth
factor (HGF) mimic having the general formula:
R -R -R -NH-(CH) -C-NH
1 2 2 n 2
where
R is an amino acid selected from the group consisting of tyrosine, phenylalanine,
aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan,
lysine, norvaline, ornithine, and s-benzyl cysteine;
R is an amino acid selected from the group consisting of tyrosine, phenylalanine,
aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan,
lysine and valine;
R is isoleucine; and
n ranges from 3-6;
and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide
bonds or reduced peptide bonds.
In a further aspect, the present invention provides N-hexanoic-L-tyrosine-L-
isoleucine-(6)-aminohexanoic amide.
In another aspect, the present invention provides a composition, comprising:
at least one hepatocyte growth factor (HGF) mimic having the general formula:
R -R -R -NH-(CH) -C-NH
1 2 3 2 n 2
1 2 3
where
R is an amino acid selected from the group consisting of tyrosine,
phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine,
methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine;
R is an amino acid selected from the group consisting of tyrosine,
phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine,
methionine, tryptophan, lysine and valine;
R is isoleucine; and
n ranges from 3-6;
and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of
peptide bonds or reduced peptide bonds; and
a carrier, said HGF mimic being dissolved or distributed in said carrier.
In another aspect, the present invention provides a composition comprising N-
hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide and a carrier, said N-hexanoic-L-
tyrosine-L-isoleucine-(6)-aminohexanoic amide being dissolved or distributed in said carrier.
In another aspect, the present invention provides use of one or more hepatocyte
growth factor mimics having the general structural formula
R -R -R -NH-(CH) -C-NH
1 2 2 n 2
where
R is an amino acid selected from the group consisting of tyrosine, phenylalanine,
aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan,
lysine, norvaline, ornithine, and s-benzyl cysteine;
R is an amino acid selected from the group consisting of tyrosine, phenylalanine,
aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan,
lysine and valine;
R is isoleucine; and
n ranges from 3-6;
and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide
bonds or reduced peptide bonds in the manufacture of a medicament for:
enhancing cognitive function or treating or preventing cognitive dysfunction in a
subject;
expanding synaptic connectivity and/or bringing about neuronal replacement in a
subject;
treating dementia;
providing neuroprotection or inducing neuroregeneration;
improving cognitive function in individuals with normal cognitive capacities thereof;
treating cancer;
treating diabetes;
treating fibrotic disease;
treating vascular insufficiency;
facilitating wound healing; or
retarding or reversing the hypervascularization of the eye.
In another aspect, the present invention provides use of N-hexanoic-L-tyrosine-L-
isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for:
enhancing cognitive function or treating or preventing cognitive dysfunction in a
subject;
expanding synaptic connectivity and/or bringing about neuronal replacement in a
subject;
treating dementia;
providing neuroprotection or inducing neuroregeneration;
improving cognitive function in individuals with normal cognitive capacities thereof;
treating cancer;
treating diabetes;
treating fibrotic disease;
treating vascular insufficiency;
facilitating wound healing; or
retarding or reversing the hypervascularization of the eye.
The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification and claims which include
the term “comprising”, other features besides the features prefaced by this term in each
statement can also be present. Related terms such as “comprise”, “comprises”, and
“comprised” are to be interpreted in similar manner.
In the description in this specification reference may be made to subject matter which
is not within the scope of the appended claims. That subject matter should be readily
identifiable by a person skilled in the art and may assist in putting into practice the invention
as defined in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A, B, and C. Effect of Dihexa on spatial learning in the water maze. A: 30
minutes before beginning testing rats were given scopolamine directly into the brain
intracerebroventricularly (ICV) and 10 minutes later Dihexa was given ICV at 10 pmoles
(low dose) or 100 pmoles (high dose). This was done daily before the first training trial.
There were 5 trials per day for 8 days. The latency to find the pedestal was considered a
measure of learning and memory. Rats receiving high Dihexa were able to completely
overcome the scopolamine deficits and were no different than controls. B. 30 minutes before
beginning testing rats were given scopolamine directly into the brain intracerebroventricularly
(ICV) and 10 minutes later Dihexa was given orally 1.25 mg/kg/day (low dose) and 2
mg/kg/day (high dose). This was done daily before the first training trial. There were 5 trials
per day for 8 days. The latency to find the pedestal was considered a measure of learning and
memory. Rats receiving high dose Dihexa were able to completely overcome the
scopolamine deficits and were no different than controls. B: Aged rats of mixed sex and age
(22-26 months) were randomly assigned to a control/untreated group or a Dihexa treated
group (2 mg/kg/day). Rats were not prescreened. Note that normally~50% of aged rats show
deficits, thus the large group errors. The Dihexa group performed significantly better than
untreated controls.
Figure 2A and B. Dihexa and Nle -AngIV dose-dependently stimulate spinogenesis. A)
Dihexa and B) Nle -AngIV increase spine density in mRFP-β-actin transfected hippocampal
neurons in a dose-dependent manner. Neurons were stimulated with Dihexa or Nle -AngIV
over a 5 day period at a wide range of concentrations. Data obtained from separate cultures;
cultures were 12 days old at time of fixing. The number of dendritic spines on representative
50 µm dendrite segments were hand counted. ** = p < 0.05 and *** = p < 0.001; n = 50;
mean ± S.E.M.; ξ = significantly different from control.
Figure 3A-E. Time dependent effects of Nle -AngIV and Dihexa treated neurons on
spinogenesis. Hippocampal neurons transfected with mRFP-β-actin were treated with 10
M Dihexa or Nle -Ang IV for 5 days in culture or for 30 minutes prior to fixation on day in
vitro 12 (DIV12), promote spinogenesis. A) Representative image of the dendritic arbor of a
day vehicle treated hippocampal neuron. B) Representative image of a dendritic arbor from
a neuron stimulated for 5 days with 10 M Dihexa. C) Representative image of the dendritic
-12 1
arbor of a neuron stimulated with 10 M Nle -Ang IV for 5 days. D) Bar graph representing
the number of spines per 50 µm dendrite length per treatment condition following a 5 day in
vitro treatment. *** P < 0.001; n = 200. E) Bar graph representing the number of spines per
50 µm dendrite length per treatment condition following an acute 30 minute treatment. *** P
< 0.001; n = 60. *Data obtained from separate cultures; cultures were 12 days old at time of
fixing. Mean ± S.E.M. by one-way ANOVA and Tukey post hoc test.
Figure 4. Nle1-AngIV and Dihexa increase spine head width. The width of the spine
head was measured as an indication of synaptic strength. Spine heads with a greater
surface area can accommodate more neurotransmitter receptors and are more likely to form
functional synapses. The AngIV analogue treatment-induced increase in spine head width
suggests facilitated neurotransmission. *** = p < 0.001; mean ± S.E.M.; n = 100.
Figure 5A-G. Neurotransmitter patterns for Nle1-AngIV and Dihexa stimulated
neurons. Dihexa and Nle1-AngIV treated neurons were immunostained for the universal
presynaptic marker synapsin and the glutamatergic presynaptic marker VGLUT1. The
percent correlation between the postsynaptic spines (red) and presynaptic puncta (green) were
measured as an indication of functional synapses. A) Bar graph representing an increase in the
number of spines following treatment with vehicle, Nle1-AngIV or Dihexa. This ensures an
active phenotype in the neurons (*** = P < 0.001; mean ± S.E.M.; n = 25). B) Bar graph
representing the percent correlation of treatment-induced postsynaptic spines to the
glutamatergic presynaptic marker VGLUT1. A high percent correlation between the
presynaptic marker and the postsynaptic spines suggests that functional connections are
formed (P > 0.05; mean ± S.E.M.; n = 25). C) Bar graph representing an increase in the
number of spines following treatment with vehicle, Nle1-AngIV or Dihexa, ensuring health
of the neurons (***=P < 0.001; mean ± S.E.M.; n = 25). D) Bar graph representing the
percent correlation of treatment-induced postsynaptic spines to the general presynaptic
marker Synapsin. No significant differences between the stimulated neurons and vehicle
control treated neurons were observed (P > 0.05; mean ± S.E.M.; n = 25) suggesting a
majority of the presynaptic input is glutamatergic. E) Bar graph representing an increase in
the number of spines following treatment with vehicle, Nle1-AngIV or Dihexa, ensuring an
active phenotype (***=P < 0.001; mean ± S.E.M.; n = 25). F) Bar graph representing the
percent correlation of treatment-induced postsynaptic spines to the postsynaptic marker PSD-
95. G) Bar graph shows no significant differences (P > 0.05; mean ± S.E.M.; n = 25)
between the postsynaptic marker PSD-95 and the postsynaptic spines suggest that the newly
formed spines have a functional postsynaptic element.
Figure 6A and B. Mini-excitatory postsynaptic currents (mEPSCs) in dissociated
hippocampal neurons. Nle1-AngIV and Dihexa treatment increase the frequency of mini-
excitatory postsynaptic currents (mEPSCs). Recordings were done on dissociated
hippocampal neurons treated with vehicle, 10 M Nle1-AngIV or Dihexa for 5 days prior to
recording. The currents recorded were spontaneous bursts of AMPA-mediated synaptic
transmission in the absence of action potentials carried in the presence of strychnine,
picrotoxin and tetrodotoxin. A) Representative traces of mEPSC recordings from Nle1-
AngIV or Dihexa treated hippocampal neurons. B) Bar graph representing the increase in
AMPA-mediated frequencies from Nle1-AngIV or Dihexa treated hippocampal neurons. The
increased frequencies indicate that spines induced by Nle1-AngIV or Dihexa support
functional synapses. *** = p < 0.001; ± S.E.M.; n = 25.
Figure 7A and B. Evaluation of Nle1-AngIV- and Dihexa-dependent spinogenesis in
CA1 hippocampal neurons from rat organotypic hippocampal slice cultures. Nle1-
AngIV- and Dihexa were found to support spinogenesis in CA1 hippocampal neurons.
Organotypic hippocampal slice cultures (400 µm thicknesses), representing a more intact
environment, were biolistically transfected with the soluble red fluorescent protein Tomato.
CA1 hippocampal neurons were selected for evaluation because of their known plastic
response during learning. Slices were obtained from postnatal day 5 rats. A) Representative
images of CA1 neuronal dendrites from Tomato transfected hippocampal slices. Images
represent a 2 day treatment with 10-12 M Nle1-AngIV or Dihexa. B) Treatment-induced
spinogenesis is observed in CA1 pyramidal hippocampal neurons. Spine numbers measured
for control slices were 7 per 50 µm dendrite length vs. 11 spines per 50µm dendrite length for
both Nle1-AngIV and Dihexa treated neurons; Mean ± S.E.M., n = 17; ** = P < 0.01
Statistical significance by one-way ANOVA followed by Tukey Multiple Comparisons Test;
Experiments were repeated at least three times.
Figure 8. HGF dose-dependently enhances spinogenesis. Effect of HGF on
spinogenesis in dissociated hippocampal neurons. Dissociated hippocampal neurons from
1 or 2 day old rats were transfected with mRFP-β-actin and stimulated with HGF for 5 days.
Treatment with 2.5 ng/ml HGF did not affect basal spine numbers and was considered sub-
threshold. Doses of 5, 10 and 20 ng/ml significantly increased the number of spines per 50
µm dendrite lengths compared to vehicle control treated neurons. *** P < 0.001; mean ±
S.E.M.; n = 50 per treatment group.
Figure 9A and B. Effects of Dihexa and HGF on spinogenesis in organotypic
hippocampal slice cultures. Hippocampal slice cultures were biolistically transfected with
the red soluble protein Tomato on DIV3 and stimulated with Dihexa or HGF on DIV5.
Organotypic hippocampal slice cultures maintain a more intact perforant path and therefore
represent a more intact environment. A) Representative images of CA1 neurons, the neuronal
type in the hippocampus that exhibits learning associated synaptic plasticity. Hippocampal
slices were stimulated with vehicle, 10 M Dihexa, or 10 ng/ml HGF for 2 days. B) Bar
graph representing the number of spines per 50 μm dendrite length for each treatment group.
Dihexa and HGF significantly increase the number of spines on CA1 hippocampal neurons
compared to control treated neurons. *** = P < 0.001; mean ± S.E.M.; n = 20 for control, 26
for Dihexa and 38 for HGF stimulated neurons.
Figure 10A-D. Effect of HGF treatment on synaptogenesis in dissociated hippocampal
neurons. HGF treatment supports the formation of functional synapses as indicated by a high
correlation between postsynaptic spines (red) and markers of presynaptic active zones
(green). A) Representative images of hippocampal neurons transfected with mRFP-β-actin
on DIV6 and treated with 10 ng/ml of HGF or vehicle for 5 days in vitro. The neurons were
stained for the general presynaptic marker Synapsin and glutamatergic presynaptic marker
VGLUT1. B) Bar graph representing an active phenotype as indicated by a significant
increase in the number of spines per 50 µm dendrite length following stimulation with HGF
(10 ng/ml). Mean number of spines = 33 vs. control = 23; *** = P < 0.001 by one-way
ANOVA and Tukey Multiple Comparisons Test; mean ± S.E.M.; n = 25). C) Percent
correlation of actin-enriched postsynaptic spines (red) juxtaposed to the universal presynaptic
marker Synapsin (green). A high percent correlation suggests functional synapses are formed.
D) Percent correlation of actin-enriched spines (red) juxtaposed to the glutamatergic
presynaptic marker VGLUT1 (green). A greater than 95% correlation suggests many of these
inputs are glutamatergic.
Figure 11. Effect of Dihexa and HGF treatment on the frequency of mEPSCs in
dissociated hippocampal neurons. Dissociated hippocampal neurons transfected with
mRFP-β-actin were stimulated with 10 M Dihexa or 10 ng/ml for 5 days prior to recording
mEPSCs. Neurons were treated with tetrodotoxin, picrotoxin, and strychnine to suppress
action potential, GABA-dependent inhibition, and glycine-dependent inhibition. Treatment
with both agonists significantly enhanced AMPA-mediated currents compared to vehicle
treated neurons (** P < 0.002; ± S.E.M. by one-way ANOVA followed by Newman-Keuls
post hoc test; n = 9, 9 and 11 respectively).
Figure 12A and B. Effect of maximal and sub-threshold doses of Angiotensin IV
analogues and HGF on spinogenesis. A) Sub-threshold levels of HGF, Dihexa or Nle1-
AngIV do not affect basal spine numbers. Combined sub-threshold levels of Dihexa (10
M) and HGF (2.5 ng/ml) phenocopy the effects of Dihexa at its biologically effective dose
alone; # = 10 M and $ = 2.5 ng/ml. B) A sub-threshold dose of the parent compound
Nle1-Ang IV (10 M) also does not affect basal spine levels. Combined sub-threshold
levels of Dihexa (10 M) and HGF (2.5 ng/ml) phenocopy the effects of Nle1-AngIV at its
biologically effective dose alone; # = 10 M and $ = 2.5 ng/ml. The ability of combined
agonists at sub-threshold doses to generate maximal responses suggests a commonality of
receptor pathways. *** P < 0.001; mean ± S.E.M.; n =50.
Figure 13A-D. The effect of the novel HGF antagonist Hinge on angiotensin IV ligand-
and HGF-mediated spinogenesis. A) The effects of the HGF antagonist Hinge (10 M) on
spinogenesis were evaluated. Hinge does not affect spinogenesis in neurons over a wide
range of doses; Dihexa was included to ensure the neurons were responsive to treatment. B)
Hinge inhibits HGF- induced spinogenesis C) Hinge inhibits Nle1-AngIV-induced
spinogenesis D) Hinge inhibits Dihexa-induced spinogenesis. # = 10 M and $ = 10 ng/ml.
The above data further indicate that the actions of Nle1-AngIV and Dihexa are mediated by
the HGF/c-Met system. *** P < 0.001; mean ± S.E.M.; n = 50.
Figure 14A-D. Effect of the HGF antagonist Hinge on HGF- and Dihexa-mediated
enhancement of mEPSCs in dissociated hippocampal neurons. Dissociated hippocampal
-12 -12
neurons were treated with Hinge (10 M), HGF, Dihexa (10 M) or HGF (10ng/ml) for 5
days after at which time mEPSCs were recorded in the absence of action potentials. A)
Representative traces of a Hinge treated neuron. B) Representative trace of a vehicle treated
neuron. C) HGF significantly augments AMPA-mediated frequencies compared to control
treated neurons. This effect is attenuated by Hinge while alone Hinge has no effect. D)
Spontaneous AMPA-mediated frequencies are significantly increased following treatment
with Dihexa and significantly reduced following pre-treatment with Hinge, which alone has
no effect on base-line frequencies. * P < 0.001; mean ± S.E.M. by one way ANOVA followed
by Newman-Keuls post hoc test.
Figure 15A-B. Distribution of c-Met protein in the adult rat brain. Gross brain regions
were obtained from adult Sprague-Dawley rats and acutely frozen in liquid nitrogen. The
samples were homogenized, separated by electrophoresis and immunoblotted for c-Met
protein and actin. A) The bar graph represents the amount of c-Met (unspecified units) in
distinct brain regions of importance to cognition. The brain samples were compared to liver
where HGF is produced. B) A representative Western blot of the samples probed against c-
Met protein (bands are at 145 kDa) and actin serving as a loading control. Equal amounts of
protein were loaded in each lane based on BCA protein determinations.
Figure 16. Stimulation of c-Met phosphorylation by HGF and Dihexa in rat
hippocampal slices. To test whether Dihexa could activate the c-Met receptor in the adult rat
brain, hippocampal slices were acutely stimulated for 30 minutes with HGF, Dihexa or
vehicle (aCSF). Receptor activation was measured by phosphorylation of the c-Met receptor
by Western blot. Saturating doses of HGF (100 ng/ml) and Dihexa (10 M) effectively
augment c-Met phosphorylation in acutely stimulated adult hippocampal slices compared to
vehicle treated slices. Sub-threshold doses of HGF (50 ng/ml) and Dihexa (10 M) did not
significantly increase c-Met receptor phosphorylation compared to control. However,
combined sub-threshold doses of HGF and Dihexa phenocopied the saturating doses of HGF
and Dihexa.
Figure 17. Effect of the HGF mimetic, Dihexa, on c-Met activation. HEK 293 cells were
treated with HGF +/- Dihexa at various doses, incubated at 37 C for 30 minutes, and then
analyzed for phosphorylated (activated) c-Met by immunoblotting. The results clearly
demonstrate the ability of HGF and Dihexa to work synergistically to activate c-Met.
Figure 18. Effect of the HGF mimetic, Dihexa, HGF-dependent cell scattering. Cell
scattering was assessed in MDCK cells. Cells were grown to confluence on coverslips,
which were then transferred to a clean plate. After treatment for four days, the number of
cells that had scattered off the coverslip was quantitated. HEX=Dihexa at 10 M.
Figure 19. Verification of c-Met receptor knockdown. Receptor knockdown was
confirmed by transfecting HEK cells with mRFP-β-actin (untransfected), a 6Myc-tagged
cMet gene product that served to verify presence of protein, shRNA (c-Met) sequences (only
sh1 was employed for the knock-down experiment) and both shRNA’s combined. The
transfected cells were cultured for a further 24 hours then lysed with RIPA buffer and
prepared for gel electrophoresis. The samples were probed against Myc by Western blot.
Untransfected cells serving as the negative control showed no signal, the 6-Myc-tagged cMet
gene product was the positive control and had a strong signal. Both the shMet1 and shMet2
sequences considerably attenuated the signal and combined did not have a signal indicating
effective knock down of the receptor.
Figure 20. Effect of c-Met knock-down on spinogenesis using a shRNA.The picture
shows a Western blot probed for Myc. Hippocampal neurons transfected with mRFP-β-actin
alone or with shMet to knock down the c-Met receptor were stimulated with HGF (10 ng/ml),
Dihexa (10-12 M) or Nle1-AngIV (10-12 M) for 48 hours. Neurons transfected with mRFP-
β-actin and stimulated with HGF, Dihexa or Nle1-AngIV significantly increased spinogenesis
(* P < 0.05; mean ± S.E.M.; n = 100). Those neurons transfected with mRFP-β-actin and
shMet did not respond to stimulation with HGF, Dihexa or Nle1-AngIV treatment,
confirming HGF and c-Met are the target (P > 0.05; mean ± S.E.M.; n = 100).
Figure 21. HGF and c-Met have a function in spatial learning and memory. The latency
to locate a submerged pedestal in the Morris water maze task of spatial learning and memory
was tested on rats to ascertain the effects of HGF/c-Met on learning and memory. Rats
received i.c.v. injections of amnestic drugs or HGF/c-Met receptor agonists. Rats treated with
the scopolamine → scopolamine are unable to learn the task as measured by latency to
escape. The group latencies for rats treated with aCSF → aCSF were significantly shorter
than the scopolamine treated group on day one of training. Scopolamine → Dihexa treated
rats and rats treated with Hinge → Hinge, while not significantly different from the
scopolamine treated group on day one of training show rapid facilitation of the task. The
group that received scopolamine + Hinge → Dihexa was not significantly different from the
scopolamine treated animals and has long latencies to escape. Group latencies to locate a
submerged pedestal in the Morris water maze task of spatial learning and memory. Hinge
alone has no effect on learning; however Hinge in addition to scopolamine prevents
facilitation of the task.
Figure 22. Stability of Norleual in rat blood as compared to D-Nle-Tyr-Ile-NH-(CH ) -
CONH .
Norleual and D-Nle-Tyr-Ile-NH-(CH ) -CONH were incubated in heparinized rat
2 5 2
blood at 37ºC; the figure shows percent recovery over time (mean ± SD). The calculated
stability t based on single phase exponential decay for Norleual was 4.6 min and for D-Nle-
Tyr-Ile-NH-(CH ) -CONH stability t was 79.97 min.
2 5 2 1/2
Figure 23. Binding of D-Nle-X-Ile-NH-(CH ) -CONH analogs to HGF. Representative
2 5 2
curves illustrating the competition of D-Nle-X-Ile-NH-(CH ) -CONH analogs for H-Hinge
2 5 2
3 -12
binding to HGF. The D-Nle-X-Ile-NH-(CH ) -CONH analogs and H-Hinge (13.3x10 M)
2 5 2
were incubated with 1.25ng of HGF for 40 min at 37 C in 0.25 ml of buffer. HGF-bound
Hinge was eluted from Bio-Gel P6 columns after the addition of different concentrations of
-13 -7
the D-Nle-X-Ile-NH-(CH ) -CONH analogs (10 -10 M). The radioactivity of the eluted
2 5 2
solutions was quantitated using scintillation counting. These data demonstrate that the D-Nle-
X-Ile-NH-(CH ) -CONH analogs exhibit a range of affinities for HGF. The K s for the Met,
2 5 2 i
-07 -09
Trp, Cys , and Tyr analogs were respectively determined to be: 1.375x10 M , 3.372x10 M,
-10 -10
1.330x10 M, and 2.426x10 M; N=9. D-Nle-Cys-Ile-NH-(CH ) -CONH , D-
2 5 2
Nle-Met-Ile-NH-(CH ) -CONH , D-Nle-Trp-Ile-NH-(CH ) - CONH , D-Nle-
2 5 2 2 5 2
Tyr-Ile-NH-(CH ) - CONH .
2 5 2
Figure 24. Inhibition of HGF dimerization by D-Nle-X-Ile-NH-(CH ) -CONH analogs.
2 5 2
HGF spontaneously dimerizes when incubated in PBS in the presence of heparin. HGF was
incubated without (control) or with various drug candidates at 10 M. These include the
derivatives of D-Nle-X-Ile- (6) amino-hexanoic amide, an AngIV-based analog family, where
X= Tyr, Cys, Trp, and Met. After 30 minute incubation, samples were cross-linked with BS3,
separated by gel electrophoresis, and silver stained. Band density was quantified and used to
determine the level of HGF dimerization in each group. Treatment groups (Tyr, Cys, Trp)
were statistically different than the HGF treated group (P<0.05; N=8) (A) Representative gel.
(B) Pooled and quantified data.
Figure 25. Inhibition of Met phosphorylation by D-Nle-X-Ile-NH-(CH ) -CONH
2 5 2
analogs. HEK293 cells were treated for 10 min with HGF+/- Nle-X-Ile-(6) amino-hexanoic
amide analogs at the indicated concentrations. HEK293 cell lysates were immunoblotted with
anti-phospho-Met and anti-Met antibodies. The differences in the mean values for Met
phosphorylation among the indicated treatment groups (Nle-X-Ile-(6) amino-hexanoic amide
analogs) compared to the HGF treated group were greater than would be expected by chance
(P <0.05; N=6). The Met group was not different than the HGF group (P>0.05; N=6).
Figure 26. Effects of D-Nle-X-Ile-NH-(CH ) -CONH analogs on MDCK cell
2 5 2
proliferation. MDCK cells were treated with a PBS vehicle (negative control), HGF, or HGF
in combination with Nle-X-Ile-(6)-amino-hexanoic amide analogs (X= L-amino acid) at 10
M concentration. The Hinge peptide (KDYIRN), which represents the dimerization domain
of HGF, was included as a positive control. The cells were allowed to grow for 4 days. Cell
numbers were estimated on the fourth day with an MTT assay by measuring absorbance at
590. % HGF-dependent proliferation: control values were subtracted from all values to
determine HGF-induced increase in cell proliferation. N=6. *** p<0.001. ** p<0.001, *
p<0.05, ns: not significant.
Figure 27. Effect of D-Nle-X-Ile-NH-(CH ) -CONH analogs on HGF-dependent
2 5 2
scattering in MDCK cells. Cell scattering in which cells lose the cell-to-cell contacts and
then migrate rapidly is the classic response to HGF. MDCK cells, the gold standard cellular
model for studying the HGF/Met system, were grown to 100% confluence on cover slips and
then placed in a clean plate. The cells were stimulated to scatter off of the cover slip by
adding 20 ng/ml of HGF to the media alone or in combination with Nle-X-Ile-(6) amino-
hexanoic amide analogs (X= L-amino acid). After 48 h of scattering, the cells were fixed with
methanol and stained with Diff-Quik. The coverslips were removed to reveal the ring of cells
that had scattered off of the cover slip and onto the plate. (A) The effect of HGF on scattering
was quantitated by determining by densitometry of the digital images from scattered cells.
ANOVA analysis indicates that the Tyr + HGF, Cys + HGF, and Trp + HGF treated groups
were different from the HGF alone group but not different from the control group. The HGF
and HGF + Met groups were not different. N=8, p<0.05 (B) Representative pictures of
MDCK cells scattering off the coverslips.
Figure 28. Correlation between inhibition of MDCK cell scattering and interference with
dimerization and the affinity to bind HGF. Three derivatives of the D-Nle-X-Ile-(6)amino-
hexanoic amide, where X is: Cys, Trp, or Met were examined to determine whether the
percent of inhibition of dimerization and the binding affinity for each compound for HGF
could be correlated to in vitro cellular activity, namely inhibition of MDCK cell scattering.
The figure shows a strong correlation between percent inhibition of HGF dimerization ( ;
R =0.9809) and for binding affinity to HGF ( ; K Values; R =0.9903) and percent
inhibition of HGF-dependent cell scattering.
Figure 29. Inhibition of B16-F10 melanoma lung colonization by D-Nle-Cys-Ile-NH-(CH ) -
CONH . 400,000 B16-F10 murine melanoma cells were injected into the tail vein of
C57BL/6 mice. Mice received daily IP injections of D-Nle-Cys-Ile-(6)-amino-hexanoic
amide (10µg/kg/day or 100µg/kg/day) or PBS vehicle.(A) After 14 days, the lungs from D-
Nle-Cys-Ile-(6)-amino-hexanoic amide treated mice exhibited an obvious reduction in
melanoma colonies when compared to untreated controls. (B) After removal, lungs were
homogenized and total melanin content was determined spectrophotometrically and used to
quantify total pulmonary melanoma colonization in vehicle treated and D-Nle-Cys-Ile-(6)-
amino-hexanoic amide treated. Ungrafted age-matched control lungs exhibited a background
absorbance at 410nm. N=15, Mean ± SEM; * P<0.05, *** P<0.001.
DETAILED DESCRIPTION
Peptide analogs or mimics of HGF (also referred to as “growth factor mimics” or
“analogs”) having a variety of therapeutic utilities have the following general structural
formula:
R -R -R -NH-(CH) -C-NH
1 2 2 n 2
where
R is an N-acyl group such as, for example, hexanoyl, heptanoyl, pentanoyl,
butanoyl, propanoyl, acetanoyl, or benzoyl,
a substituted or unsubsituted phenyl,
a D or L norleucine,
an amino acid (D or L) such as, for example, lysine, arginine, norvaline,
ornithine, or S-benzyl cysteine amino acid residues;
R is an amino acid (D or L), such as, for example, tyrosine, cysteine, phenyalanine,
aspartic acid, glutamic acid, glycine, tryptophan, lysine, homocysteine, homoserine,
homophenylalanine;
R is a D or L isoleucine, leucine or valine amino acid residue; and
n ranges from 3-6;
and wherein covalent bonds 1, 2 and 3 are either peptide bonds (e.g. -CO-NH- or reduced
peptide bonds (CH -NH ).
An exemplary peptide bond and reduced peptide bond are depicted below:
Peptide bond Reduced peptide bond
Compounds within the general structural formula have been synthesized and analyzed
according to the following procedures.
Standard synthesis method:
All compounds were synthesized by solid phase methods using an AAPPTEC
Endeavor 90 peptide synthesizer using Fmoc protected amino acids. All peptide amides were
synthesized on a Rink resin. The resin was pre-swollen in dimethylformamide (DMF) and
deprotected with 20% piperidine/ DMF for 30 minutes. The piperidine/DMF was then
removed by filtration. After deprotection, the N-α Fmoc protected amino acid was added to
reaction vessel as a dry powder (3 equivalents). The vessel was then filled with 2/3 full with
DMF and dry diisopropylethylamine (DIPEA; 3.5-4 equivalents) was added. Next N-[(1H-
benzotriazolyl)(dimethylamino)methylene]-N-methyl-methanaminium
hexafluorophosphate N-oxide (HBTU; 2.9 equivalents) was added and the suspension mixed
for 30 minutes. The solution was then removed by filtration. The resin was then washed twice
with DMF, twice with methanol, twice with dichloromethane, and finally twice more with
DMF. Solutions were removed by filtration after each wash. Coupling efficiency was
monitored using a Kaiser test for free amines. If the test was positive the amino acid was re-
coupled to the resin or growing peptide chain. If the test indicated a good linkage, the resin
was washed once more with DMF, deprotected with 20% piperidine/ DMF for 30 minutes as
indicated above, and again washed with DMF. The coupling then proceeded as indicated
above.
Acylation of the N-terminal of the peptide:
After final deprotection, the peptide resin is incubated with 20% of the appropriate
acyl anhydride in DMF and DIPEA (1.5 equivalents) for 30 minutes at room temperature. The
resin was now washed twice with DMF, twice with methanol, twice with dichloromethane,
and finally twice more with DMF. The solution was removed by filtration and a Kaiser test
was performed to verify the completeness of the capping. If free amine was detected the
capping procedure was repeated.
Insertion of an N-terminal reduced peptide bond:
After deprotection, hexanal (3 equivalents) DMF was added to the resin and allowed
to mix for 5 minutes. Next, 3 equivalents of sodium cyanoborohydride were added and the
suspension was mixed for an additional 2 hours. After the standard washing procedure was
performed (see above), the Kaiser test was again used to verify the completeness of the
reaction. If coupling was deemed incomplete, the procedure was repeated.
Cleavage of peptide from Rink Resin:
After the last amino acid was deprotected and washed the resin was transferred to a
sintered glass funnel (4 porosity) and the DMF removed by vacuum. The semi-dry resin was
then suspended in 20% trifluoroacetic acid (TFA) with 2.5% triisopropyl-silane as a
scavenger, incubated at room temperature for 15 minutes, and filtered. The resin was washed
three times with additional DMF and filtered. Ten volumes of ice-cold diethyl ether were
added to the combined filtrates and the mixture allowed to set at 4 C overnight. Precipitated
peptide was recovered by filtration and washed three times with ice-cold ether. For very
hydrophobic peptides the combined ether washes were re-extracted with DMF, allowed to
precipitate peptide, and filtered to recover additional peptide.
Peptide purification and analysis:
Crude peptides were first purified by reverse phase HPLC using a C18 column using
gradient elution. The typical gradient was 10% to 40% component B over 30 minutes at a
flow rate of 1 ml/min at 37 C where component A was 80 mM triethyamine phosphate, pH
3.0 and component B was acetonitrile (ACN). In all instances only a single peak with 215nm
absorption was detected and collected. The collected compound was lyophilized and
redissolved in 20% methanol and injected onto a second C18 column. . The HPLC/MS
system used was from Shimadzu (Kyoto, Japan), consisting of a CBM-20A communications
bus module, LC-20AD pumps, SIL-20AC auto sampler, SPD-M20A diode array detector and
LCMS-2010EV mass spectrometer. Data collection and integration were achieved using
Shimadzu LCMS solution software. The analytical column used was an Econosphere C18
(100mm x 2.1mm) from Grace Davison Discovery Science (Deerfield, IL, USA). The mobile
phase consisted of HPLC grade methanol and water with 0.1% trifluoroacetic acid.
Separation was carried out using a non-isocratic method (20% - 50% methanol over 30 min)
at 37 C and a flow rate of 0.3 mL/min. For MS analysis, a positive ion mode (Scan) was used
and peaks analyzed at the anticipated m/z. Typical peak purity analysis revealed a peak purity
index of >0.95. Wavelength ratioing with the diode array detector further confirmed peak
purity.
Table 1 below presents a listing of compounds in Family 1, drawn to mimetics, and
Famillies 2-5, drawn to antagonists, all of which have been synthesized and analyzed
according to the procedures described above.
TABLE 1
General Structure of Family 1 (Mimetics) and Families 2-5 (Antagonists)
R -R -R -NH-(CH) -C-NH
1 2 2 n 2
Arrows 1-3 denote pb = peptide bond; ψ = reduced peptide bond (CH -NH )
Family # R (N-acyl group) R2 R3 1
1 hexanoyl Tyr Ile pb
heptanoyl Tyr Ile pb
pentanoyl Tyr Ile pb
butanoyl Tyr Ile pb
propanoyl Tyr Ile pb
acetanoyl Tyr Ile pb
benzoyl Tyr Ile pb
hexanoyl Tyr Ile ψ
Family # R1 R2 R3
2 D-Nle Tyr Ile
D-Nle Phe Ile
D-Nle Asp Ile
D-Nle Arg Ile
D-Nle Ile Ile
D-Nle Ser Ile
D-Nle His Ile
D-Nle Gly Ile
D-Nle Cys Ile
D-Nle Met Ile
D-Nle Trp Ile
D-Nle Lys Ile
D-Nle Val Ile
D-Nle Gly D-Ile
R1 R2 R3
3 D-Nle D-Tyr Ile
D-Nle D-Phe Ile
D-Nle D-Asp Ile
D-Nle D-Arg Ile
D-Nle D-Ile Ile
D-Nle D-Ser Ile
D-Nle D-His Ile
D-Nle D-Gly Ile
D-Nle D-Cys Ile
D-Nle D-Met Ile
D-Nle D-Trp Ile
D-Nle D-Lys Ile
R1 R2 R3
4 Tyr Tyr Ile
Phe Tyr Ile
Asp Tyr Ile
Arg Tyr Ile
Ile Tyr Ile
Ser Tyr Ile
His Tyr Ile
Gly Tyr Ile
Cys Tyr Ile
Met Tyr Ile
Typ Tyr Ile
Lys Tyr Ile
R1 R2 R3
D-Tyr Tyr Ile
D-Phe Tyr Ile
D-Asp Tyr Ile
D-Arg Tyr Ile
D-Ile Tyr Ile
D-Ser Tyr Ile
D-His Tyr Ile
D-Cys Tyr Ile
D-Met Tyr Ile
D-Typ Tyr Ile
D-Lys Tyr Ile
With reference to Table 1, while a number of compounds which have been
synthesized include tyrosine and isoleucine at R and R , respectively, a wide range of amino
acid and other residues might be used for the mimetics or agonists (Family 1 and Families 2-
, respectively) in the practice of embodiments of the invention or described herein at these
other positions including, without limitation, tyrosine, cysteine, methionine, phenylalaine,
aspartic acid, glutamic acid, histidine, tryptophan, lysine, leucine, valine, homocysteine,
homoserine, and homophenyalanine. Further, while the mimetics include certain N-acyl
groups as specified in Table 1 (Family 1), in the practice of various embodiments of the
invention or described herein other N-acyl groups or substituted or unsubstituted phenyl
groups may be used at R . In addition, while a number of the agonists in Table 1 (Families 2-
) have norleucine at R , or an amino acid residue, in the practice of various embodiments of
this invention a number of an amino acid residues (D or L) may be used at residue R ,
including without limitation, tyrosine, phenylalanine, aspartic acid, arginine, isoleucine,
serine, histidine, glycine, cysteine, methionine, tryptophan, norvaline, ornithine, S-benzyl
cysteine amino acid residues. Finally, while all the compounds synthesized and tested in
Table 1 included 5 methyl repeats, the methyl repeats (n) could range from 3-6 within the
practice of the some of the embodiments of the present invention.
Compounds within Table 1 have also been assessed as follows:
Assessment of HGF mimetic activity:
HGF mimetic activity was typically assessed by one or both of two methods:
augmentation of HGF-dependent c-Met phosphorylation in HEK293 cells, or 2) augmentation
of HGF-dependent cell scattering in MDCK cells. All the compounds in Family one were
tested using the c-Met phosphorylation assay. N- hexanoyl-Tyr-Ile-(6) aminohexamide was
further evaluated and found to have spectacularly augment HGF-dependent MDCK cell
scattering. Table 2 presents a summary of the results.
TABLE 2
Compound ( 10 M) HGF Mimetic Activity
N- heptanoyl-Tyr-Ile-(6) aminohexamide ++++
N- hexanoyl-Tyr-Ile-(6) aminohexamide ++++
N- pentaanoyl-Tyr-Ile-(6) aminohexamide ++++
N- butanoyl-Tyr-Ile-(6) aminohexamide +++
N- propananoyl-Tyr-Ile-(6) aminohexamide ++
N- acetanoyl-Tyr-Ile-(6) aminohexamide +
N- benzoyl-Tyr-Ile-(6) aminohexamide +
N- hexanoyl-ψ (CH -NH )-Tyr-Ile-(6) aminohexamide +++
Cell culture. Human embryonic kidney cells 293 (HEK293), Madin Darby canine kidney
cells (MDCK), and B16F10 murine melanoma cells were grown in DMEM, 10% fetal bovine
serum (FBS). Cells were grown to 90-100% confluency before use. For most but not all
studies HEK and MDCK cells were serum starved for 24 hours prior to the initiation of drug
treatment.
Western blotting. HEK293 cells were seeded in 6 well tissue culture plates and grown to
95% confluency in DMEM containing 10% FBS. The cells were serum deprived for 24 hours
prior to the treatment to reduce the basal levels of phospho-Met. Following serum starvation,
cocktails comprised of vehicle and HGF (2.5 ng/ml) with/without the test compound were
prepared and pre-incubated for 30 minutes at room temperature. The cocktail was then added
to the cells for 10 minutes to stimulate the Met receptor and downstream proteins. Cells were
harvested using RIPA lysis buffer (Upstate) fortified with phosphatase inhibitor cocktails 1
and 2 (Sigma-Aldrich; St. Louis, MO). The lysate was clarified by centrifugation at 15,000 g
for 15 minutes, protein concentrations were determined using the BCA total protein assay,
and then appropriate volumes of the lysates were diluted with 2x reducing Laemmli buffer
and heated for ten minutes at 95° C. Samples containing identical amounts of protein were
resolved using SDS-PAGE (Criterion, BioRad Laboratories), transferred to nitrocellulose,
and blocked in Tris-buffered saline (TBS) containing 5% milk for one hour at room
temperature. The phospho-Met antibody was added to the blocking buffer at a final
concentration of 1:1000 and incubated at 4° C overnight with gentle agitation. The
membranes were then washed several times with water and TBS (PBS, 0.05% Tween-20), a
1:5000 dilution of horseradish-peroxidase conjugated goat anti-rabbit antiserum was added,
and the membranes further incubated for one hour at room temperature. Proteins were
visualized using the Supersignal West Pico Chemiluminescent Substrate system (Pierce,
Fenton, MO) and molecular weights determined by comparison to protein ladders
(BenchMark, Invitrogen; and Kaleidoscope, BioRad). Images were digitized and analyzed
using a UVP phosphoimager.
Scattering assay. MDCK cells were grown to 100% confluency on the coverslips in six-well
plates and washed twice with PBS. The confluent coverslips were then aseptically transferred
to new six well plates containing 900 µl serum free DMEM. Norleual, Hinge peptide, and/or
HGF (2.5 ng/ml) were added to appropriate wells. Control wells received PBS vehicle.
Plates were incubated at 37°C with 5% CO for 48 hours. Media was removed and cells were
fixed with methanol. Cells were stained with Diff-Quik Wright-Giemsa (Dade-Behring,
Newark, DE) and digital images were taken. Coverslips were removed with forceps and
more digital images were captured. Pixel quantification of images was achieved using Image
J and statistics were performed using Prism 5 and InStat v.3.05.
For the general structural formula presented above, and reproduced below for ease of
reference, there are several different compounds which can be prepared according to the
synthesis procedures described above and used for therapies described below. Table 3
identifies various exemplary families with various listed compounds in those families
(identified by substitution of moieties within the general formula).
TABLE 3
General Structure:
R -R -R -NH-(CH) -C-NH
1 2 3 2 n 2
1 2 3
Arrows 1-3 may be pb = peptide bond; ψ = reduced peptide bond (CH -NH )
Family # R R2 R3 n 1 2 3
1 hexanoyl Y I 5 pb pb pb
heptanoyl Y I 5 pb pb pb
pentanoyl Y I 5 pb pb pb
butanoyl Y I 5 pb pb pb
propanoyl Y I 5 pb pb pb
acetanoyl Y I 5 pb pb pb
isopropanoyl Y I 5 pb pb pb
tert-butanoyl Y I 5 pb pb pb
isobutanoyl Y I 5 pb pb pb
benzoyl Y I 5 pb pb pb
2 hexanoyl Y I 5 ψ pb pb
heptanoyl Y I 5 ψ pb pb
pentanoyl Y I 5 ψ pb pb
butanoyl Y I 5 ψ pb pb
propanoyl Y I 5 ψ pb pb
acetanoyl Y I 5 ψ pb pb
isopropanoyl Y I 5 ψ pb pb
tert-butanoyl Y I 5 ψ pb pb
isobutanoyl Y I 5 ψ pb pb
benzoyl Y I 5 ψ pb pb
3 hexanoyl Y I 5 ψ pb ψ
heptanoyl Y I 5 ψ pb ψ
pentanoyl Y I 5 ψ pb ψ
butanoyl Y I 5 ψ pb ψ
propanoyl Y I 5 ψ pb ψ
acetanoyl Y I 5 ψ pb ψ
isopropanoyl Y I 5 ψ pb ψ
tert-butanoyl Y I 5 ψ pb ψ
isobutanoyl Y I 5 ψ pb ψ
benzoyl Y I 5 ψ pb ψ
4 hexanoyl Y I 5 pb pb ψ
heptanoyl Y I 5 pb pb ψ
pentanoyl Y I 5 pb pb ψ
butanoyl Y I 5 pb pb ψ
propanoyl Y I 5 pb pb ψ
acetanoyl Y I 5 pb pb ψ
isopropanoyl Y I 5 pb pb ψ
tert-butanoyl Y I 5 pb pb ψ
isobutanoyl Y I 5 pb pb ψ
benzoyl Y I 5 pb pb ψ
5 hexanoyl F I 5 pb pb pb
heptanoyl F I 5 pb pb pb
pentanoyl F I 5 pb pb pb
butanoyl F I 5 pb pb pb
propanoyl F I 5 pb pb pb
acetanoyl F I 5 pb pb pb
isopropanoyl F I 5 pb pb pb
tert-butanoyl F I 5 pb pb pb
isobutanoyl F I 5 pb pb pb
benzoyl F I 5 pb pb pb
6 hexanoyl F I 5 ψ pb pb
heptanoyl F I 5 ψ pb pb
pentanoyl F I 5 ψ pb pb
butanoyl F I 5 ψ pb pb
propanoyl F I 5 ψ pb pb
acetanoyl F I 5 ψ pb pb
isopropanoyl F I 5 ψ pb pb
tert-butanoyl F I 5 ψ pb pb
isobutanoyl F I 5 ψ pb pb
benzoyl F I 5 ψ pb pb
7 hexanoyl F I 5 ψ pb ψ
heptanoyl F I 5 ψ pb ψ
pentanoyl F I 5 ψ pb ψ
butanoyl F I 5 ψ pb ψ
propanoyl F I 5 ψ pb ψ
acetanoyl F I 5 ψ pb ψ
isopropanoyl F I 5 ψ pb ψ
tert-butanoyl F I 5 ψ pb ψ
isobutanoyl F I 5 ψ pb ψ
benzoyl F I 5 ψ pb ψ
8 hexanoyl F I 5 pb pb ψ
heptanoyl F I 5 pb pb ψ
pentanoyl F I 5 pb pb ψ
butanoyl F I 5 pb pb ψ
propanoyl F I 5 pb pb ψ
acetanoyl F I 5 pb pb ψ
isopropanoyl F I 5 pb pb ψ
tert-butanoyl F I 5 pb pb ψ
isobutanoyl F I 5 pb pb ψ
benzoyl F I 5 pb pb ψ
9 hexanoyl C I 5 pb pb pb
heptanoyl C I 5 pb pb pb
pentanoyl C I 5 pb pb pb
butanoyl C I 5 pb pb pb
propanoyl C I 5 pb pb pb
acetanoyl C I 5 pb pb pb
isopropanoyl C I 5 pb pb pb
tert-butanoyl C I 5 pb pb pb
isobutanoyl C I 5 pb pb pb
benzoyl C I 5 pb pb pb
hexanoyl C I 5 ψ pb pb
heptanoyl C I 5 ψ pb pb
pentanoyl C I 5 ψ pb pb
butanoyl C I 5 ψ pb pb
propanoyl C I 5 ψ pb pb
acetanoyl C I 5 ψ pb pb
isopropanoyl C I 5 ψ pb pb
tert-butanoyl C I 5 ψ pb pb
isobutanoyl C I 5 ψ pb pb
benzoyl C I 5 ψ pb pb
11 hexanoyl C I 5 ψ pb ψ
heptanoyl C I 5 ψ pb ψ
pentanoyl C I 5 ψ pb ψ
butanoyl C I 5 ψ pb ψ
propanoyl C I 5 ψ pb ψ
acetanoyl C I 5 ψ pb ψ
isopropanoyl C I 5 ψ pb ψ
tert-butanoyl C I 5 ψ pb ψ
isobutanoyl C I 5 ψ pb ψ
benzoyl C I 5 ψ pb ψ
12 hexanoyl C I 5 pb pb ψ
heptanoyl C I 5 pb pb ψ
pentanoyl C I 5 pb pb ψ
butanoyl C I 5 pb pb ψ
propanoyl C I 5 pb pb ψ
acetanoyl C I 5 pb pb ψ
isopropanoyl C I 5 pb pb ψ
tert-butanoyl C I 5 pb pb ψ
isobutanoyl C I 5 pb pb ψ
benzoyl C I 5 pb pb ψ
13-16 Same pattern as families 1-4 with R2=S
17-20 Same pattern as families 1-4 with R2=T
21-24 Same pattern as families 1-4 with R2=D
-28 Same pattern as families 1-4 with R2=E
29-32 Same pattern as families 1-4 with R2=Y, R3=V
33-36 Same pattern as families 1-4 with R2=F, R3=V
37-40 Same pattern as families 1-4 with R2=C, R3=V
41-44 Same pattern as families 1-4 with R2=S, R3=V
45-48 Same pattern as families 1-4 with R2=T, R3=V
49-52 Same pattern as families 1-4 with R2=D, R3=V
53-56 Same pattern as families 1-4 with R2=E, R3=V
57-85 Same pattern as families 29-56 with R3=L
86-170 Same pattern as families 1-85 with n=3
171-256 Same pattern as families 1-85 with n=4
257-341 Same pattern as families 1-85 with n=6
R R R n 1 2 3
1 2 3
342 D-norleucine Y I 5 pb pb pb
D-norleucine F I 5 pb pb pb
D-norleucine C I 5 pb pb pb
D-norleucine S I 5 pb pb pb
D-norleucine T I 5 pb pb pb
D-norleucine D I 5 pb pb pb
D-norleucine E I 5 pb pb pb
D-norleucine G I 5 pb pb pb
343 D-norleucine Y I 5 pb pb ψ
D-norleucine F I 5 pb pb ψ
D-norleucine C I 5 pb pb ψ
D-norleucine S I 5 pb pb ψ
D-norleucine T I 5 pb pb ψ
D-norleucine D I 5 pb pb ψ
D-norleucine E I 5 pb pb ψ
D-norleucine G I 5 pb pb ψ
344 D-norleucine Y I 5 ψ pb pb
D-norleucine F I 5 ψ pb pb
D-norleucine C I 5 ψ pb pb
D-norleucine S I 5 ψ pb pb
D-norleucine T I 5 ψ pb pb
D-norleucine D I 5 ψ pb pb
D-norleucine E I 5 ψ pb pb
D-norleucine G I 5 ψ pb pb
345 D-norleucine Y I 5 ψ pb ψ
D-norleucine F I 5 ψ pb ψ
D-norleucine C I 5 ψ pb ψ
D-norleucine S I 5 ψ pb ψ
D-norleucine T I 5 ψ pb ψ
D-norleucine D I 5 ψ pb ψ
D-norleucine E I 5 ψ pb ψ
D-norleucine G I 5 ψ pb ψ
346-349 Same pattern as families 342-345 with R3=V
350-353 Same pattern as families 342-345 with R3=L
354-365 Same pattern as families 342-353 with R1=D norvaline
366-377 Same pattern as families 342-345 with R3=D-lysine
378-389 Same pattern as families 342-345 with R3=D-arginine
390-401 Same pattern as families 342-345 with R3=D S-methyl cysteine
402-457 Same pattern as families 342-401 with n=3
458-513 Same pattern as families 342-401 with n=4
514-569 Same pattern as families 342-401 with n=6
Alternatively, the analogs or growth factor mimics of the present invention or
described herein may also be represented as comprised of four elements joined by covalent
peptide or reduced peptide bonds, as follows:
I - II - III - IV
where
I = an acid such as heptanoic, hexanoic, pentanoic, butyric, proprionic, acetic, benzoic, or
substituted benzoic acid, and isoforms thereof; or D or L norleucine, lysine, arginine,
norvaline, ornithine, or S-benzyl cysteine
II = a D or L cysteine, phenyalanine, aspartic acid, glutamic acid, serine, tyrosine, glycine,
homocysteine, homoserine or homophenylalanine amino acid residue;
III = a D or L isoleucine, leucine, or valine amino acid residue; and
IV = amino-hexanoic, amino-pentanoic or amino butyric acid; wherein elements I, II, III and
IV are joined by peptide or reduced peptide bonds.
In one embodiment, the analog is: hexanoic-tyrosine-isoleucine-(6)-amino-hexanoic
amide. Using Formula I as a generic formula, for this particular analog, R1= hexanoyl; R2 is
Tyr; R3 is Ile; and n = 5. Alternatively, using the I - II - III - IV nomenclature, in this
embodiment, I = hexanoic acid, II = Tyr; III = Ile; and IV = hexanoic amide.
Embodiments of the invention involve providing one or more HGF mimics to a
subject in need thereof. Exemplary subjects or patients which might benefit from receiving
therapy such as administration of the one or more HGF mimics described herein are generally
mammals, and usually humans, although this need not always be the case, since veterinary
and research related applications of the technology are also contemplated. Generally a
suitable subject or patient in need of therapy are identified by, for example, a health care
professional or professionals using known tests, measurements or criteria. For example, in
the treatment for dementia, a subjects already having symptoms of dementia, or being at risk
of developing symptoms of dementia will be identified. Similar identification processes will
be followed for other diseases and/or disorders (e.g., cancer therapy, other cognitive
dysfunction therapies, etc.). A suitable treatment protocol is then developed based on the
patient, the disease and/or disorder and its stage of development, and the HGF mimic and its
dosage and delivery format, as well as other relevant factors. The subject then receives
treatment with HGF mimic. Embodiments of the invention also comprise one or more steps
related to monitoring the effects or outcome of administration in order to evaluate the
treatment protocol and/or to adjust the protocol as required or in a manner that is likely to
provide more benefit, e.g. by increasing or decreasing doses of medication, or by changing the
particular type of mimic that is administered, or by changing the frequency of dosing or the
route of administration, etc. With particular reference to the embodiment of providing
cognitive enhancement for example, while in some cases the improvement in cognition (or
the prevention of loss of cognition) that occurs may be complete, e.g. the functioning of the
patient returns to or remains normal (as assessed in comparison to suitable control subjects or
standardized values obtained therefrom), this need not always be the case. Those of skill in
the art will recognize that even a lower level of improvement in cognition may be highly
beneficial to the patient, as may be the slowing of the progression of a disease, as opposed to
a complete cure.
The methods described herein involve administering compositions comprising the
HGF mimics disclosed herein to a patient in need thereof. The present invention thus also
provides compositions which comprise the HGF analogs/mimics of the present invention,
usually together with a pharmacologically suitable carrier or diluent. In some embodiments,
one substantially purified HGF mimic is present in a composition; in other embodiments
more than one HGF mimic is present, each HGF mimic being substantially purified prior to
being mixed in the composition. The preparation of pharmacologically suitable compositions
for use as medicaments is well known to those of skill in the art. Typically, such
compositions are prepared either as liquid solutions or suspensions, however solid forms such
as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution
in, or suspension in, liquids prior to administration may also be prepared. The preparation
may also be emulsified. The active ingredients may be mixed with excipients which are
pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients
are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations
thereof. In addition, the composition may contain minor amounts of auxiliary substances such
as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to
administer an oral form of the composition, various thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders and the like may be added. The composition of the
present invention may contain any such additional ingredients so as to provide the
composition in a form suitable for administration. The final amount of HGF mimic in the
formulations may vary. However, in general, the amount in the formulations will be from
about 1% to about 99%.
The HGF mimic compositions (preparations) of the present invention may be
administered by any of the many suitable means which are well known to those of skill in the
art, including but not limited to: by injection, inhalation, orally, intravaginally, intranasally,
by ingestion of a food or product containing the mimic, topically, as eye drops, via sprays,
etc. In preferred embodiments, the mode of administration is orally or by injection. In
addition, the compositions may be administered in conjunction with other treatment
modalities such as other agents which are used to treat, for example, dementia or the
conditions which cause dementia in the patient, examples of which include but are not limited
to the administration of anti-depressants and psychoactive drugs, administration of dopamine
and similar agents. Similarly, in cancer treatment modalities, the HGF mimics may be
administered together with analgesics and other suitable drugs. Thus, in embodiments of the
invention, one or more HGF mimics may be used in combination with one or more different
bioactive drugs.
The amount of HGF inhibitor that is administered may be in the range of from about
0.1 to about 1,000 mg/kg, an preferably in the range of from about 1 to about 100mg/kg,
although as one of skill in the art will recognize, the precise amount may vary depending on
one or more attributes of the drug recipient, including but not limited to: weight, overall
health, gender, age, nationality, genetic history, other conditions being treated, etc., and larger
or smaller doses are within the practice of this invention. Dosing may also take place
periodically over a period of time, and the dosage may change (increase or decrease) with
time.
The HGF mimics of the invention may be used to treat a variety of cognitive function
disorders (cognitive dysfunction) as well as other disorders that are related to HGF activity or
lack thereof. “Cognitive function” or “cognition” as used herein refers to a range of high-level
brain functions, including but not limited to: the ability to learn and remember information;
the ability to organize, plan, and problem-solve; the ability to focus, maintain, and shift
attention as necessary; and to understand and use language; the ability to accurately perceive
the environment; the ability to perform calculations. Such functions include but are not
limited to memory (e.g. acquiring, retaining, and retrieving new information); attention and
concentration (particularly divided attention); information processing (e.g. dealing with
information gathered by the five senses); executive functions (e.g. planning and prioritizing);
visuospatial functions (e.g. visual perception and constructional abilities); verbal fluency and
speech (e.g. word-finding); general intellect (e.g. “intelligence”); long-term (remote) memory;
conversational skills; reading comprehension; etc. Conversely, by “cognitive dysfunction”
we mean the loss of such abilities. Losses may be measured, detected and/or diagnosed in any
of the many ways known to those of ordinary skill in the art. Such methods include but are
not limited to: the use of standardized testing administered by a professional (puzzles, word
games or problems, etc.); by self-reporting and/or the reports of caretakers, friends and family
members of an afflicted individual; by observation of the activities, life skills, habits and
coping mechanisms of the individual by professional or lay persons; by the results of
questionnaires administered to an afflicted individual; etc.
Such disorders may be caused, for example, by a decrease in synaptic connectivity
and/or neuron density due to a variety of factors. In some embodiments, the loss is caused by
a brain injury, e.g. traumatic brain injury. Traumatic brain injury, which is occurring at record
levels as a result of wars and sporting activities, is characterized by reduced neuronal
connectivity. Hence, the use of HGF mimetics represents a viable treatment option. Such
brain injuries may be the result of an external trauma to the brain, e.g. caused by a high
impact accident (e.g. a car accident, a fall, etc.), a shooting incident, a sports injury (e.g.
caused by impact to the head such a boxers and football players experience); injuries received
in combat, etc. Alternatively, such injuries may be the result of internal brain trauma, e.g. as
the result of stroke, aneurism, surgical procedure, tumor, etc. or other types of conditions
which result in lack of oxygen to the brain or to sections of the brain; injuries due to
inhalation of toxic gases; due to aging of the brain; to diseases and disorders which exert a
deleterious effect on the nervous system and/or brain, such as multiple sclerosis, Parkinson’s
disease, Huntington’s disease, brain disorders such as schizophrenia, etc.
As a specific example of a therapy contemplated by embodiments of the invention, the
HGF mimics may be used for the treatment of dementia. By “dementia” we mean a serious
loss of cognitive ability in a previously unimpaired person, beyond what might be expected
from normal aging. It may be static, the result of a unique global brain injury, or progressive,
resulting in long-term decline due to damage or disease in the body. Although dementia is far
more common in the geriatric population, it may occur in any stage of adulthood. For the
purposes of embodiments of this invention, the term “dementia” may include and/or be
caused by e.g. Alzheimer's disease, vascular dementia, dementia with Lewy bodies, etc. or
combinations of these. In other embodiments of the invention, Alzheimer's disease may be
excluded from this definition. Other causes of dementia which may be treated as described
herein include but are not limited to hypothyroidism and normal pressure hydrocephalus.
Inherited forms of the diseases which cause or are associated with dementia that may treated
as described herein include but are not limited to: frontotemporal lobar degeneration,
Huntington's disease, vascular dementia, dementia pugilistica, etc. In younger populations,
progressive cognitive disturbance may be caused by psychiatric illness, alcohol or other drug
abuse, or metabolic disturbances. Certain genetic disorders can cause true neurodegenerative
dementia in younger populations (e.g. 45 and under). These include familial Alzheimer's
disease, SCA17 (dominant inheritance); adrenoleukodystrophy (X-linked); Gaucher's disease
type 3, metachromatic leukodystrophy, Niemann-Pick disease type C, pantothenate kinase-
associated neurodegeneration, Tay-Sachs disease and Wilson's disease. Vitamin deficiencies
and chronic infections may also occasionally mimic degenerative dementia. These include
deficiencies of vitamin B12, folate or niacin, and infective causes including cryptococcal
meningitis, HIV, Lyme disease, progressive multifocal leukoencephalopathy, subacute
sclerosing panencephalitis, syphilis and Whipple's disease. With respect to rapidly
progressive dementia, Creutzfeldt-Jakob disease typically causes a dementia which worsens
over weeks to months, being caused by prions. The common causes of slowly progressive
dementia also sometimes present with rapid progression, e.g. Alzheimer's disease, dementia
with Lewy bodies, and frontotemporal lobar degeneration (including corticobasal
degeneration and progressive supranuclear palsy).
In addition, encephalopathy or delirium may develop relatively slowly and result in
dementia. Possible causes include brain infection (viral encephalitis, subacute sclerosing
panencephalitis, Whipple's disease) or inflammation (limbic encephalitis, Hashimoto's
encephalopathy, cerebral vasculitis); tumors such as lymphoma or glioma; drug toxicity (e.g.
anticonvulsant drugs); metabolic causes such as liver failure or kidney failure; and chronic
subdural hematoma. The dementia that is treated according to methods described herein may
also be the result of other conditions or illnesses. For example, there are many medical and
neurological conditions in which dementia only occurs late in the illness, or as a minor
feature. For example, a proportion of patients with Parkinson's disease develop dementia,
Cognitive impairment also occurs in the Parkinson-plus syndromes of progressive
supranuclear palsy and corticobasal degeneration (and the same underlying pathology may
cause the clinical syndromes of frontotemporal lobar degeneration). Chronic inflammatory
conditions of the brain may affect cognition in the long term, including Behçet's disease,
multiple sclerosis, sarcoidosis, Sjögren's syndrome and systemic lupus erythematosus.
In addition, inherited conditions may also cause dementia alongside other features include:
Alexander disease, Canavan disease, cerebrotendinous xanthomatosis, fragile X-associated
tremor/ataxia syndrome, glutaric aciduria type 1, Krabbe's disease, maple syrup urine disease,
Niemann Pick disease type C, Kufs' disease, neuroacanthocytosis, organic acidemias,
Pelizaeus-Merzbacher disease, urea cycle disorders, Sanfilippo syndrome type B, and
spinocerebellar ataxia type 2.
In addition to treating dementia, the HGF mimics of the invention may be used for
neuroprotection and/or to treat neurodegenerative diseases, some of which also involve
dementia as described above. For neuroprotection, the HGF mimics may be administered
propylactically, i.e. prior to a subject’s encounter with or exposure to a potential neurohazard.
For example, the mimics may be administered prior to exposure to a drug, chemical or
medical procedure that is known or likely to cause neuronal damage. With respect to the
treatment of neurodegenerative diseases, the general pro-survival anti-apoptotic activity of
HGF supports the use of HGF mimetics for treating neurodegenerative diseases including but
not limited to Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis
(ALS), etc.
In addition, the mimics may be used for the treatment of “depression”, by which we
mean major depressive disorder (MDD) (also known as recurrent depressive disorder, clinical
depression, major depression, unipolar depression, or unipolar disorder) and also depression
that is characteristic of bipolar disorder, etc. Depression is ultimately a disease in which
neurons and synaptic contacts are lost in the hippocampus. The capacity of HGF to induce
new synaptic connections and stimulate neurogenesis in the hippocampus supports the use of
HGF mimetics for the treatment of depression.
In addition, the cognitive abilities of persons afflicted with certain genetic
predispositions to cognitive dysfunction may also be increased, e.g. persons with genetic
disorders such as Down’s syndrome, lack of proper brain development e.g. due to lack of
oxygen before or during birth, various congenital disorders which interfere with brain
development, etc.
As demonstrated in the Examples below, the HGF mimics can inhibit the HGF/Met
system, and therefore can be used as anti-cancer agents. The HGF mimics may be used to
attenuate malignant and metastatic transformations.
The HGF mimics have application in the therapy of Fibrotic Disease. Hepatic, renal,
cardiac, and pulmonary fibrosis is a growing problem in our aging population. Unfortunately,
the degradation of function that accompanies fibrotic changes is difficult to treat. The
dramatic ability of HGF to inhibit or reverse tissue fibrosis suggests that orally-active HGF
mimics provides a therapeutic option.
The HGF mimics have application in the therapy of Peripheral Vascular Disease:
Lower Extremity Arterial Disease. Vascular disease resulting in poor perfusion is a common
sequel of diabetes, obesity, and atherosclerosis. One treatment option is the induction of new
collateral vessels in the effected organs and tissues. The potent angiogenic activity of HGF
and HGF mimics can provide a clinical utility for the treatment of vascular insufficiency.
HGF mimics may also be used for Wound Healing. Defective wound healing is a
hallmark of diabetics and burn victims. The ability of HGF to promote wound healing
because of its angiogenic and mitogenic activities supports the use of HGF mimics to enhance
the wound healing process. Data indicates that several HGF mimics are effective wound
repair enhancers in both normal and diabetic individuals.
Without being bound by theory, it is believed that the likely mechanism underlying
this marked pro-cognitive activity is augmented synaptic connectivity. This is likely due to an
increase in miniature synaptic activity brought about by increasing dendritic spine densities
and altering the morphological phenotype of postsynaptic spines.
The foregoing Examples are provided in order to illustrate various embodiments of
the invention, but should not be interpreted as limiting the invention in any way.
EXAMPLES
EXAMPLE 1. Regulation of Synaptogenesis by Dihexa and Nle1-AngIV.
The tetrapeptide (Nle1-YIH) and tripeptide (Nle1-YI) fragments of the Nle1-AngIV
analog of AngIV were previously found to be the smallest active fragments capable of
overcoming scopolamine-induced cognitive dysfunction in a spatial learning task. Using the
tripeptide as a new template, additional active analogues were synthesized with improved
metabolic stability, blood brain barrier permeability, and oral activity. In this Example, we
show the characterization of the novel, orally active, angiotensin IV analogue Dihexa.
MATERIALS AND METHODS
Animals and Surgery. Male Sprague-Dawley rats (Taconic derived) weighing 390-450 g
were maintained with free access to water and food (Harland Tekland F6 rodent diet,
Madison, WI) except the night prior to surgery when food was removed. Each animal was
anesthetized with Ketamine hydrochloride plus Xylazine (100 and 2 mg/kg im. respectively;
Phoenix Scientific; St. Joseph, MO, and Moby; Shawnee, KS). An intracerebroventricular
(icv) guide cannula (PE-60, Clay Adams; Parsippany, NY) was stereotaxically positioned
(Model 900, David Kopf Instruments; Tujunga, CA) in the right hemisphere using flat skull
coordinates 1.0 mm posterior and 1.5 mm lateral to bregma (refer to Wright et al. 1985). The
guide cannula measured 2.5 cm in overall length and was prepared with a heat bulge placed
2.5 mm from its beveled tip, thus acting as a stop to control the depth of penetration. Once in
position, the cannula was secured to the skull with two stainless-steel screws and dental
cement. Post-operatively the animals were housed individually in an American Accreditation
for Laboratory Animal Care-approved vivarium maintained at 22±1° C on a 12-h alternating
light/dark cycle initiated at 06:00 h. All animals were hand gentled for 5 min per day during
the 5-6 days of post-surgical recovery. Histological verification of cannula placement was
accomplished by the injection of 5 µl fast-green dye via the guide cannula following the
completion of behavioral testing. Correct cannula placement was evident in all rats utilized in
this study.
Behavioral testing. The water maze consisted of a circular tank painted black (diameter: 1.6
m; height: 0.6 m), filled to a depth of 26 cm with 26-28° C water. A black circular platform
(diameter: 12 cm; height: 24 cm) was placed 30 cm from the wall and submerged 2 cm below
the water surface. The maze was operationally sectioned into four equal quadrants designated
NW, NE, SW, and SE. For each rat the location of the platform was randomly assigned to
one of the quadrants and remained fixed throughout the duration of training. Entry points
were at the quadrant corners (i.e. N, S, E, and W) and were pseudo-randomly assigned such
that each trial began at a different entry point than the preceding trial. Three of the four
testing room walls were covered with extra-maze spatial cues consisting of different shapes
(circles, squares, triangles) and colors. The swimming path of the animals was recorded
using a computerized video tracking system (Chromotrack; San Diego Instruments, CA). The
computer displayed total swim latency and swim distance. Swim speed was determined from
these values.
Each member of the treatment groups in the scopolamine studies received an icv injection
of scopolamine hydrobromide (70 nmol in 2 µl aCSF over a duration of 20 s) 30 min prior to
testing followed by Dihexa 10 min prior to testing. Control groups received scopolamine or
aCSF 20 min prior to testing followed by aCSF 10 min prior testing. The behavioral testing
protocol has been described previously in detail (Wright et al. 1999). The rats in the aged rat
study on received Dihexa of aCSF (control group).Briefly, acquisition trials were conducted
on 8 consecutive days with 5 trials/day. On the first day of training the animal was placed on
the platform for 30 s prior to the first trial. Trials commenced with the placement of the rat
facing the wall of the maze at one of the assigned entry points. The rat was allowed a
maximum of 120 s to locate the platform. Once the animal located the platform it was
permitted a 30 s rest period on the platform. If the rat did not find the platform, the
experimenter placed the animal on the platform for the 30 s rest period. The next trial
commenced immediately following the rest period.
Following day 8 of acquisition training, one additional trial was conducted during which
the platform was removed (probe trial). The animal was required to swim the entire 120 s to
determine the persistence of the learned response. Total time spent within the target quadrant
where the platform had been located during acquisition and the number of crossings of that
quadrant was recorded. Upon completion of each daily set of trials the animal was towel-
dried and placed under a 100 watt lamp for 10-15 min and then returned to its home cage.
Hippocampal cell culture preparation. Hippocampal neurons (2x10 cells per square cm)
were cultured from P1 Sprague Dawley rats on plates coated with poly-L-lysine from Sigma
(St..Louis, MO; molecular weight 300,000). Hippocampal neurons were maintained in
Neurobasal A media from Invitrogen (Carlsbad, CA) supplemented with B27 from
Invitrogen, 0.5 mM L-glutamine, and 5mM cytosine-D-arabinofuranoside from Sigma added
at 2 days in vitro. Hippocampal neurons were then cultured a further 3–7 days, at which time
they were either transfected or treated with various pharmacological reagents as described in
(Wayman, Davare et al. 2008).
Transfection. Neurons were transfected with mRFP-β-actin on day in vitro 6 (DIV6) using
LipofectAMINE ™ 2000 (Invitrogen) according to the manufacturer’s protocol. This protocol
yielded the desired 3-5% transfection efficiency thus enabling the visualization of individual
neurons. Higher efficiencies obscured the dendritic arbor of individual neurons. Expression
of fluorescently tagged actin allowed clear visualization of dendritic spines, as dendritic
spines are enriched in actin. On DIV7 the cells were treated with vehicle (H 0) or peptides
(as described in the text) added to media. On DIV12 the neurons were fixed (4%
paraformaldehyde, 3% sucrose, 60 mM PIPES, 25 mM HEPES, 5 mM EGTA, 1 mM MgCl ,
pH 7.4) for 20 min at room temperature and mounted. Slides were dried for at least 20 hours
at 4°C and fluorescent images were obtained with Slidebook 4.2 Digital Microscopy Software
driving an Olympus IX81 inverted confocal microscope with a 60X oil immersion lens, NA
1.4 and resolution 0.280 µm Dendritic spine density was measured on primary and secondary
dendrites at a distance of at least 150 µm from the soma. Five 50 µm long segments of
dendrite from at least 10 neurons per data point were analyzed for each data point reported.
Each experiment was repeated at least three times using independent culture preparations.
Dendrite length was determined using the National Institutes of Health’s Image J 1.41o
program (NIH, Bethesda, MD) and the neurite tracing program Neuron J (Meijering, Jacob et
al. 2004) Spines were manually counted.
Organotypic Hippocampal Slice Culture Preparation and Transfection. Hippocampi from P4
Sprague Dawley rats were cultured as previously described (Wayman, Impey et al. 2006).
Briefly, 400 µm slices were cultured on (Milipore, Billerica, MA) for 3 days after which they
were biolistically transfected with tomato fluorescent protein (TFP) using a Helios Gen Gun
(BioRad, Hercules, CA), according to the manufacturer’s protocol, to visualize dendritic
arbors. Following a 24 hour recovery period slices were stimulated with vehicle (H O), 1pM
Nle1-AngIV or Dihexa for 2 days. Slices were fixed and mounted. Hippocampal CA1
neuronal processes were imaged and measured as described above.
Immunocytochemistry. Transfected neurons were treated, fixed and stained. Briefly, cells
were permeablized with 0.1% Triton X-100 detergent (Bio-Rad; Hercules, CA) for 10
minutes. An 8% bovine serum albumin (Intergen Company; Burlington, MA) in PBS was
used to prevent non-specific binding for one hour at R.T.; Primary antibody incubations were
at a 1:2500 dilution (see below) in 1% BSA in PBS at 4°C overnight. Secondary antibody,
1:3000 Alexafluor 488 goat-anti-mouse (Invitrogen: Carlsbad, CA) was applied for two hours
at room temperature. Coverslips were mounted with ProLong Gold anti-fade reagent
(Invitrogen; Carlsbad, CA) and all washes were done with PBS. Imaging and analysis were
performed as described above. For presynaptic excitatory transmission the VGLUT1
(Synaptic Systems, Goettingen, Germany) marker (Balschun, Moechars et al.) was employed
and for general presynaptic transmission synapsin1 (Synaptic Systems, Goettingen, Germany)
(Ferreira and Rapoport 2002) was applied. A postsynaptic function was established by PSD-
95 (Milipore, Billerica, MA) (El-Husseini, Schnell et al. 2000). In each instance the total
number of spines was counted for the treatment groups, control, Nle1-AngIV and Dihexa, to
ensure an active phenotype. The total number of actin enriched spines adjacent to VGLUT1
or Synapsin were counted and converted to a percentage as the percent correlation of
treatment-induced spines to presynaptic markers is a strong indicator of ability to transmit
excitatory signals. In our application the number of correlations consisted of red fluorescent-
tagged actin spines against green PSD-95 immunopositive puncta which, when merged,
resulted in an orange spine.
Whole-cell recordings. Patch-clamp experiments were performed on mRFP-β-actin
transfected cultured hippocampal neurons (vehicle control) and on transfected hippocampal
neurons with 1pM Nle1-AngIV or Dihexa 5 day pretreatment. Recordings were taken from
neurons that were pyramidal-like in shape (~20 µm cell bodies and asymmetric dendrite
distribution). The time after transfection was 6 days. The culture medium was exchanged by
an extracellular solution containing (in mM) 140 NaCl, 2.5 KCl, 1 MgCl , 3 CaCl , 25
glucose, and 5 HEPES; pH was adjusted to 7.3 with KOH; osmolality was adjusted to 310
mOsm. Cultures were allowed to equilibrate in a recording chamber mounted on inverted
microscope (IX-71; Olympus optical, Tokyo) for 30 min before recording. Transfected cells
were visualized with fluorescence (Olympus optical). Recording pipettes were pulled (P-97
Flaming/Brown micropipette puller; Sutter Instrument, Novato, CA) from standard-wall
borosilicate glass without filament (OD = 1.5 mm; Sutter Instrument). The pipette-to-bath DC
resistance of patch electrodes ranged from 4.0 to 5.2MΩ, and were filled with a internal
solution of the following composition (in mM): 25 CsCl, 100 CsCH O S, 10
phosphocreatine, 0.4 EGTA, 10 HEPES, 2 MgCl , 0.4 Mg-ATP, and 0.04 Na-GTP; pH was
adjusted to 7.2 with CsOH; osmolality was adjusted to 296 - 300 mOsm. Miniature EPSCs
(mEPSCs) were isolated pharmacologically by blocking GABA receptor chloride channels
with picrotoxin (100 µM; Sigma), blocking glycine receptors with strychnine (1 µM; Sigma),
and blocking action potential generation with tetrodotoxin (TTX, 500 nM; Tocris).
Recordings were obtained using a Multiclamp 700B amplifier (Molecular Devices,
Sunnyvale, CA). Analog signals were low-pass Bessel filtered at 2 kHz, digitized at 10 kHz
through a Digidata 1440A interface (Molecular Devices), and stored in a computer using
Clampex 10.2 software (Molecular Devices). The membrane potential was held at -70 mV at
room temperature (25ºC) during a period of 0.5 – 2 h after removal of the culture from the
incubator. Liquid junction potentials were not corrected. Data analysis was performed using
Clampfit 10.2 software (Molecular Devices), and Mini-Analysis 6.0 software (Synaptosoft
Inc.; Fort Lee, NJ). The criteria for successful recording included the electrical resistance of
the seal between the outside surface of the recording pipette and the attached cell >2 GΩ,
neuron input resistance >240 MΩ. The mEPSCs had a 5-min recording time.
RESULTS
Nle1-AngIV has long been known to be a potent cognitive enhancing agent (Wright
and Harding, 2008 ) but is limited in terms of clinical utility by its metabolic instability
(t =1.40 minutes in rat serum). In order to exploit the pro-cognitve properties of AngIV like
molecules more metabolically stable analogs needed to be developed. As part of this
development process Dihexa (N-hexanoic-Tyr-Ile-(6)-aminohexanoic amide) was synthesized
and characterized (t =330minutes in rat serum). To determine if the stabilized analog,
Dihexa still possessed pro-cognitive/ anti-dementia activity it was tested in two dementia
models- the scolpolamine amnesia and the aged rat models. These studies demonstrated that
Dihexa was able to reverse the cognitive deficits observed in both models. Dihexa delivered
either intracerebroventricularly or orally by gavage improved water maze performance
reaching performance levels seen in young healthy rats. In Figure 1A Dihexa delivered at 100
pmoles (n=8, p<.01)but not 10 pmoles reversed scopolamine-dependent learning deficits as
evidenced by an escape latency equivalent to non-scopolamine treated controls. Similar
results were seen when Dihexa was delivered orally (Figure 1B) at both low (1.25mg/kg/day)
and high (2mg/kg/day). The high dose group’s performance was no different than controls
(n=8, p<.01). Randomly grouped aged rats ( 20-24 weeks) included both sexes were similarly
treated with oral Dihexa over the 8 day test period (n=8) and compared to untreated controls
(Figure 1C). The results indicate that the treated rats preformed significantly better in the
water maze than untreated rats. (p<.05).
One hypothesis that was put forward to explain the pro-cognitive effects of Nle1-
AngIV and Dihexa was that they were acting as hepatocyte growth factor mimetics and as
such may be supporting he expansion of neuronal connectivity by inducing the growth of
dendritic spines and the establishment of numerous new synapses. To determine the influence
of Dihexa on spinogenesis and synaptogenesis in high density mRFP-β-actin transfected
hippocampal neuronal cultures was assayed. Actin-enriched spines increased in response to
Dihexa and Nle1-AngIV treatment in a dose-dependent manner (Figure 2A and B). An
apparent ceiling effect was produced by 10 M Dihexa application (mean ± S.E.M.; 30
spines per 50 µm dendrite length vs. 19 for control; *** = P < 0.001; n = 50 and 100
respectively) while the results of a 10 M dose were not significantly different from control
treated neurons (mean ± S.E.M.; 21 spines per 50 µm dendrite for both groups vs. 19 for
control; * = P < 0.05; n = 95 and 100 respectively). They were however statistically different
-12 -10
from the 10 M Dihexa dose. Neurons receiving a 10 M dose of Dihexa had fewer spines
than vehicle treated neurons (Mean ± S.E.M.; 11 spines per 50 µm dendrite length vs. 19 for
control; # = P < 0.01; n = 50 and 100 respectively). Nle1-AngIV similarly induced a dose-
dependent increase is spine density with a marked difference in the 10 M dose which
promoted spinogenesis (mean ± S.E.M.; 22 spines per 50 µm dendrite length vs. 17 for
control; ** = P < 0.01; n = 50). Maximal increases in spine density were again observed
following treatment with a 10 M dose (mean ± S.E.M.; 25 and 26 spines per 50 µm
dendrite length respectively vs. 17 for control; ** = P < 0.01; n = 50). The 10 M dose of
Nle1-AngIV also had no effect on basal spine numbers (mean ± S.E.M.; 17 spines per 50 µm
dendrite length vs. 17 for control; ** = P < 0.01; n = 50).
The effects of a long-term application (5 days) of the AT4 agonists Dihexa and Nle1-
AngIV were compared to an acute application of the agonists (30 minutes) at the biologically
effective dose of 10 M (Figure 3A-E). The results revealed a near 3-fold increase in the
number of spines stimulated by Dihexa and greater than 2-fold increase for Nle1-AngIV
stimulated spines following a 5 day treatment (Figure 3 D). Both treatment groups differed
significantly from the vehicle control group for which the average number of spines per 50
µm dendrite length was 15. The average number of spines for the Dihexa and Nle1-AngIV
treated groups was 41 and 32 spines per 50 µm dendrite lengths, respectively (mean ± S.E.M.,
n = 200; *** = P < 0.001 by one-way ANOVA and Tukey post hoc test). The behavioral data
(data not shown) suggest a quick mechanism of action is taking place during acquisition of
the spatial memory task. Therefore the ability of both Dihexa and Nle1-AngIV to promote
spinogenesis was measured by an acute 30 minute application on the final day of culturing
(Figure 3 E). The acute 30 minute application of Dihexa and Nle1-AngIV, on the 12th day
in vitro (DIV12) reveals a significant increase in spines compared to 30 minute vehicle
treated neurons (Dihexa mean spine numbers per 50 µm dendrite length = 23.9 ± S.E.M.;
Nle1-AngIV mean spine numbers = 2.6 ± S.E.M.; mean spine numbers for vehicle control
treated neurons = 17.4 ± S.E.M.; n = 60; *** = p < 0.0001 by one-way ANOVA followed by
Tukey post-hoc test).
Strong correlations exist between spine size, persistence of spines, number of AMPA-
receptors and synaptic efficacy. A correlation between the existence of long-term memories
to spine volume has also been suggested (Kasai, Fukuda et al., 2001; Yasumatsu, Matsuzaki
et al. 2008). With these considerations in mind spine head size measurements were taken.
Results indicate that 10 M doses of Dihexa and Nle1-AngIV increased spine head width
(Figure 4). Average spine head width for Nle1-AngIV = 0.87 μm (*** = P < 0.001; mean ±
S.E.M.) and Dihexa = 0.80 μm (** = P < 0.01; mean ± S.E.M.) respectively compared to
control head size (0.67 μm).
Dihexa and Nle1-AngIV Mediate Synaptogenesis
To quantify synaptic transmission, mRFP-β-actin transfected neurons were immuno-
stained against synaptic markers. Hippocampal neurons were stimulated for 5 days in vitro
with 10 M Dihexa or Nle1-AngIV (Figure 5A-F). Nle1-AngIV and Dihexa’s
neurotransmitter patterns were probed for excitatory synaptic transmission by staining against
the glutamatergic presynaptic marker Vesicular Glutamate Transporter 1 (VGLUT1)
(Balschun, Moechars et al. 2010). The universal presynaptic marker Synapsin was employed
to measure juxtaposition of the newly formed spines with presynaptic boutons (Ferreira and
Rapoport 2002). PSD-95 served as a marker for the postsynaptic density (El Husseini,
Schnell et al. 2000).
Dihexa and Nle1-AngIV treated neurons significantly augmented spinogenesis; mean
spine numbers per 50 µm dendrite length for Nle1-AngIV = 39.4; mean spine numbers per 50
µm dendrite length for Dihexa = 44.2; mean spine numbers per 50 µm dendrite length for
vehicle treated neurons = 23.1 (mean ± S.E.M., *** = P < 0.001) (Figure 3B, D and F and
Table 4). The percent correlation for the newly formed spines to the synaptic markers was
calculated as a measure for the formation of functional synapses. Dihexa and Nle1-AngIV
treatment-induced spines did not differ from control treated neurons in percent correlation to
VGLUT1, Synapsin or PSD-95 (P > 0.05) (Figure 5A, C and E and Table 4).
Table 4. Summary of the percent correlation to markers of synaptic components and the
number of spines induced by Dihexa and Nle1-AngIV treatment.
Treatment Control Nle1-AngIV Dihexa
Number of spines/50µm 22 39 44
% Correlation VGLUT1 95.2 95.1 94.4
Number of spines/50µm 19 31 37
% Correlation Synapsin 93.4 94.2 96.3
Number of spines/50µm 18 36 43
% Correlation PSD-95 98.03 97.38 98.71
The total number of spines for each treatment group is indicated as the number of spines per
50 µm dendrite length. The percent correlation of the presynaptic marker Synapsin, the
glutamatergic presynaptic marker VGLUT1 or the postsynaptic component PSD-95 is
reported directly below. N = 25 for each treatment group.
The above results suggest that the newly formed dendritic spines produced by Dihexa
and Nle1-AngIV treatment are creating functional synapses. To further support this
conclusion, mini postsynaptic excitatory currents (mEPSCs), the frequency of which
corresponds to the number of functional synapses were recorded from mRFP-β-actin
transfected hippocampal neurons. A near two-fold increase in the AMPA-mediated currents
was measured following treatment with 10-12 M Nle1-AngIV and Dihexa (Figure 6A and
B). The mean frequency of AMPA-mediated mEPSCs recorded from vehicle treated neurons
was 3.06 ± 0.23 Hz from 33 cells. Nle1-AngIV induced a 1.7 fold increase over percent
control frequency (5.27 ± 0.43 Hz from 25 cells; Mean ± S.E.M.; *** = P < 0.001 vs. control
group and Dihexa produced a 1.6 fold increase (4.82 ± 0.34 Hz from 29 cells; *** = P <
0.001 vs. control group confirming an amplification of functional synapses. No differences in
amplitude, rise- or decay-times were observed (data not shown) which suggests that the
individual properties of the synapse were not altered.
To further assess the physiological significance of the spine induction witnessed in
dissociated neonatal hippocampal neurons the effects of Dihexa and Nle1-AngIV on spine
formation in organotypic hippocampal slice cultures was evaluated. These preparations, while
still neonatal in origin, represent a more intact and three dimensional environment than
dissociated neurons. Hippocampal CA1 neurons, which have been functionally linked to
hippocampal plasticity and learning/memory, could be easily identified based on morphology
and were singled out for analysis. Dihexa and Nle1-AngIV significantly augmented
spinogenesis in organotypic hippocampal slice cultures when compared to vehicle treated
neurons. There were no differences in spine numbers between the Dihexa and Nle1-AngIV
treatment groups (Figure 7A and B). Spine numbers measured for control slices were 7 per
50 µm dendrite length vs. 11 spines per 50 µm dendrite length for both Nle1-AngIV and
Dihexa treated neurons; mean ± S.E.M., n = 13-20; ** = P < 0.01.
DISCUSSION
In this study, Dihexa like Nle1-AngIV was a potent cognitive enhancer when given
either ICV or orally. As predicted, Dihexa and Nle1-AngIV both promoted spinogenesis and
enhance synaptogenesis in cultured rat hippocampal neurons. As expected of an angiotensin
IV analogue, Dihexa exerted spine induction effects at sub-nano-molar concentrations
(Harding, Cook et al. 1992; Krebs, Hanesworth et al. 2000) with some spine formation by
Dihexa and Nle1-AngIV occurring as early as 30 minutes after stimulation (Figure 3D). The
maximal effect, however, requires a significantly longer treatment period (Figure 3C).
Spine head size measurements were taken as an indicator of synaptic potentiation.
Larger spines with a greater surface area tend to have larger synapses, a larger PSD to recruit
scaffolding proteins, and a greater number of glutamatergic receptive neurotransmitter
receptors (Kennedy 1997). Although not different from one another (P > 0.05), both Dihexa
and Nle1-AngIV treatment groups exhibited large expansions in spine head size. Changes in
spine morphology and numbers are proposed to be mechanisms for converting short-term
synaptic changes into highly stable and long-lasting changes (Hering and Sheng 2001).
To evaluate the functional significance of these spine changes Nle1-AngIV and
Dihexa stimulated hippocampal neurons were immunostained against the glutamatergic
presynaptic marker VGLUT1 (Balschun, Moechars et al. 2010), the general presynaptic
marker Synapsin (Ferreira and Rapoport 2002) and the postsynaptic marker PSD-95
(Kennedy 1997; Han and Kim 2008) to decipher neurotransmitter phenotypes. The high and
unaltered correlation between VGLUT1, Synapsin, and PSD-95 in both treated and control
dendrites suggests that the newly minted spines support functional synapses (Figure 5 and
Table 4) (Han and Kim 2008; Yasumatsu, Matsuzaki et al. 2008). Further, a near perfect
correlation between mRFP-β-actin labeled spines and the general presynaptic marker
Synapsin and VLGUT1 staining, which identifies excitatory glutamatergic synapses suggests
that most AngIV-dependent effects on hippocampal spines were restricted to excitatory
synapses. These findings correspond nicely with the findings of De Bundel et al. in which no
effect on the inhibitory neurotransmitter GABA by native angiotensin IV was observed (De
Bundel, Demaegdt et al. 2010).
The increase in mEPSC frequency observed by Dihexa and Nle1-AngIV treated
preparations further supports that new spines form functional synapses (Malgaroli and Tsien
1992; Hering and Sheng 2001; Tyler and Pozzo-Miller 2003). The consistent strengthening
of neurotransmission initiated by Dihexa and Nle1-AngIV could not be attributed to intrinsic
fluctuations of neurotransmitter release or metabolic and mechanical influences (Yasumatsu,
Matsuzaki et al. 2008). The data presented here suggest that Nle1-AngIV and Dihexa
increase miniature synaptic activity by increasing dendritic spine densities and altering the
morphological phenotype of postsynaptic spines in-vitro and may represent the mechanism
that underlies facilitated learning observed AngIV analogues (Wright, Stubley et al. 1999;
Lee, Albiston et al. 2004).
To bridge the adult behavioral data to the in vitro mechanistic theory, organotypic
hippocampal slice cultures that maintain an environment representative of an intact
hippocampus were employed and evaluated for treatment-induced spinogenesis. Application
of 10 M Ne1-AngIV and Dihexa in ballistically transfected hippocampal slices significantly
increase spine densities (Figure 7) implying that such changes may in fact be occurring in the
intact hippocampus.
Thus, Dihexa fits the criteria necessary for an effective anti-dementia drug: 1) it is
orally active, as it survives passage through the gut and enters the brain; 2) it augments
neuronal connectivity, a necessary property when faced with loss of neuronal connectivity;
and 3) it is inexpensive to synthesize thus making it accessible to patients.
EXAMPLE 2. The Target of AngIV Analogs is Hepatocyte Growth Factor
This Example shows that the novel angiotensin IV ligand Dihexa and its parent
molecule Nle1-AngIV act through the HGF/c-Met receptor system.
MATERIALS AND METHODS
Animals and Surgery
Male Sprague-Dawley rats (Taconic derived) weighing 390-450 g were maintained
with free access to water and food (Harland Tekland F6 rodent diet, Madison, WI) except the
night prior to surgery when food was removed. Each animal was anesthetized with Ketamine
hydrochloride plus Xylazine (100 and 2 mg/kg im. respectively; Phoenix Scientific; St.
Joseph, MO, and Moby; Shawnee, KS). An intracerebroventricular (icv) guide cannula (PE-
60, Clay Adams; Parsippany, NY) was stereotaxically positioned (Model 900, David Kopf
Instruments; Tujunga, CA) in the right hemisphere using flat skull coordinates 1.0 mm
posterior and 1.5 mm lateral to bregma (Wright et al., 1985). The guide cannula measured
2.5 cm in overall length and was prepared with a heat bulge placed 2.5 mm from its beveled
tip, thus acting as a stop to control the depth of penetration. Once in position, the cannula
was secured to the skull with two stainless-steel screws and dental cement. Post-operatively
the animals were housed individually in an American Accreditation for Laboratory Animal
Care-approved vivarium maintained at 22±1° C on a 12-h alternating light/dark cycle initiated
at 06:00 h. All animals were hand gentled for 5 min per day during the 5-6 days of post-
surgical recovery.
Behavioral Testing
The water maze consisted of a circular tank painted black (diameter: 1.6 m; height:
0.6 m), filled to a depth of 26 cm with 26-28° C water. A black circular platform (diameter:
12 cm; height: 24 cm) was placed 30 cm from the wall and submerged 2 cm below the water
surface. The maze was operationally sectioned into four equal quadrants designated NW, NE,
SW, and SE. For each rat the location of the platform was randomly assigned to one of the
quadrants and remained fixed throughout the duration of training. Entry points were at the
quadrant corners (i.e. N, S, E, W) and were pseudo-randomly assigned such that each trial
began at a different entry point than the preceding trial. Three of the four testing room walls
were covered with extra-maze spatial cues consisting of different shapes (circles, squares,
triangles) and colors. The swimming path of the animals was recorded using a computerized
video tracking system (Chromotrack; San Diego Instruments, CA). The computer displayed
total swim latency and swim distance. Swim speed was determined from these values.
Each member of the treatment groups received an icv injection of scopolamine
hydrobromide (70 nmol in 2 µl aCSF over a duration of 20 s) 20 min prior to testing followed
by Dihexa (300 pmol in 2 µl aCSF), Hinge (300 pmol in 2 µl aCSF), or Hinge + Dihexa (300
pmol in 4 µl aCSF) 5 min prior to testing. This scopolamine preparation is a generally
accepted animal model of the spatial memory dysfunction that accompanies dementia (Fisher
et al., 2003). Control groups received scopolamine or aCSF 20 min prior to testing followed
by aCSF 5 min prior testing. The behavioral testing protocol has been described previously in
detail (Wright et al., 1999). Briefly, acquisition trials were conducted on 8 consecutive days,
trials/day. On the first day of training the animal was placed on the pedestal for 30 s prior to
the first trial. Trials commenced with the placement of the rat facing the wall of the maze at
one of the assigned entry points. The rat was allowed a maximum of 120 s to locate the
platform. Once the animal located the platform it was permitted a 30 s rest period on the
platform.
If the rat did not find the platform, the experimenter placed the animal on the platform
for the 30 s rest period. The next trial commenced immediately following the rest period.
Upon completion of each daily set of trials the animal was towel-dried and placed under a
100 watt lamp for 10-15 min and then returned to its home cage.
Statistical Analyses
One-way ANOVA was used to analyze the dendritic spine results and significant
effects were analyzed by Tukey post-hoc test. Morris water maze data set mean latencies to
find the platform during each daily block of five trials were calculated for each animal for
each day of acquisition. One-way ANOVAs were used to compare group latencies on Days
1, 4, and 8 of training. Significant effects were analyzed by Newman-Keuls post-hoc test
with a level of significance set at P < 0.05.
Scattering assay. MDCK cells were grown to 100% confluency on the coverslips in six-well
plates and washed twice with PBS. The confluent coverslips were then aseptically transferred
to new six well plates containing 900 µl serum free DMEM. Norleual, Hinge peptide, and/or
HGF (20 ng/ml) were added to appropriate wells. Control wells received PBS vehicle. Plates
were incubated at 37°C with 5% CO for 48 hours. Media was removed and cells were fixed
with methanol. Cells were stained with Diff-Quik Wright-Giemsa (Dade-Behring, Newark,
DE) and digital images were taken. Coverslips were removed with forceps and more digital
images were captured. Pixel quantification of images was achieved using Image J and
statistics were performed using Prism 5 and InStat v.3.05.
Dissociated Hippocampal Neuronal cell culture preparation
Hippocampal neurons (2x10 cells per square centimeter) were cultured from P1–2
Sprague Dawley rats on plates coated with poly-L-lysine from Sigma (St..Louis, MO;
molecular weight 300,000). Hippocampal neurons were maintained in Neurobasal A media
from Invitrogen (Carlsbad, CA) supplemented with B27 from Invitrogen, 0.5 mM L-
glutamine, and 5mM cytosine-D-arabinofuranoside from Sigma added at 2 days in vitro.
Hippocampal neurons were then cultured a further 3–7 days, at which time they were either
transfected or treated with various pharmacological reagents as described in the text or figure
legends.
Transfection of Dissociated Hippocampal Neuronal Cell Cultures
Neurons were transfected with mRFP-β-actin on day in vitro 6 (DIV6) using
LipofectAMINE ™ 2000 (Invitrogen) according to the manufacturer’s protocol. This
protocol yielded the desired 3-5% transfection efficiency thus enabling the visualization of
individual neurons. Higher efficiencies obscured the dendritic arbor of individual neurons.
Expression of fluorescently tagged actin allowed clear visualization of dendritic spines, as
dendritic spines are enriched in actin. On DIV7 the cells were treated with vehicle (H20) or
peptides (as described in the text) added to media. On DIV12 the neurons were fixed (4%
paraformaldehyde, 3% sucrose, 60 mM PIPES, 25 mM HEPES, 5 mM EGTA, 1 mM MgCl ,
pH 7.4) for 20 min at room temperature and mounted. Slides were dried for at least 20 hours
at 4°C and fluorescent images were obtained with Slidebook 4.2 Digital Microscopy Software
driving an Olympus IX81 inverted confocal microscope with a 60X oil immersion lens, NA
1.4 and resolution 0.280 µm Dendritic spine density was measured on primary and secondary
dendrites at a distance of at least 150 µm from the soma. Five 50 µm long segments of
dendrite from at least 10 neurons per data point were analyzed for each data point reported.
Each experiment was repeated at least three times using independent culture preparations.
Dendrite length was determined using the National Institutes of Health’s Image J 1.41o
program (NIH, Bethesda, MD) and the neurite tracing program Neuron J (Meijering, Jacob et
al. 2004) Spines were manually counted.
Organotypic Hippocampal Slice Culture Preparation and Transfection
Hippocampi from P4 Sprague Dawley rats were cultured as previously described
(Wayman, Impey et al. 2006). Briefly, 400 µm slices were cultured on (Milipore, Billerica,
MA) for 3 days after which they were biolistically transfected with tomato fluorescent protein
(TFP) using a Helios Gene Gun (BioRad, Hercules, CA), according to the manufacturer’s
protocol, to visualize dendritic arbors. Following a 24 hour recovery period slices were
stimulated with 1pM Nle1-AngIV or Dihexa for 2 days. Slices were fixed and mounted.
Hippocampal CA1 neuronal processes were imaged and measured as described above.
Acute Hippocampal Slices
Adult Sprague-Dawley rats (250g +) obtained from Harlan Laboratories (Ca, USA)
were anesthetized with isofluorane (Vet OneTM, MWI, Meridian, ID, USA) and decapitated.
The brain was rapidly removed and placed into ice-chilled artificial cerebrospinal fluid
(aCSF) for approximately 30 s. Both hemispheres were separated by a mid-saggital cut and
both hippocampi removed. Slices were sectioned cross- and length-wise (400 μm) to ensure
penetrability of the drug, using a McIlwain tissue chopper (Brinkmann, Gomshall, UK) and
transferred to a gassed (95% O /5% CO ) incubation chamber containing aCSF for 90
minutes at room temperature. Slices were transferred to fresh tubes, aCSF was removed by
careful suctioning and replaced with aCSF containing vehicle (aCSF + aCSF), 100 ng/ml with
carrier free adult recombinant Hepatocyte Growth Factor (HGF) (R and D Systems, MN,
-10 -10
USA) in aCSF, 10 M Hinge (Harding lab), 50 ng/ml in aCSF, 10 M Dihexa (Harding
-12 -12
lab) in aCSF, 10 M Dihexa in aCSF or 50 ng/ml HGF + 10 M Dihexa in aCSF for 30
minutes at 37°C with gentle rocking. aCSF was removed and the slices were lysed using
RIPA buffer (Upstate/Milipore, Billerica, MA) and inhibitor Cocktails I and II (Sigma,
St.Louis, MO), sonicated on ice and clarified by centrifugation for 30 minutes, 13,000 rpm at
4°C. The supernatant was removed from the pellet and stored at -80°C or processed
immediately for gel electrophoresis.
shRNA
A target sequence for c-Met was designed using RNAi central design program (see the
website located at cancan.cshl.edu/). The target sequence
GTGTCAGGAGGTGTTTGGAAAG (SEQ ID NO: 2) was inserted into pSUPER vector
(Oligoengine, Seattle WA) which drives endogenous production of shRNA under the H1
promoter. The shRNA was transfected into cells using the lipofectamine method described
above. Verification of receptor knockdown was done by creating a c-MetMyc tagged gene
product using the Gateway cloning system (Invitrogen). The Met protein coding sequence
was cloned from rat whole brain cDNA using primers obtained from Integrated DNA
Technologies, Inc. The amplified product was gel purified and a band corresponding to 190
kDa band excised and cloned into a PCAGGSMyc destination vector (Gateway).
Gel Electrophoresis and Western Blotting
Protein concentration of the samples was quantified using the BCA method (Pierce,
Rockford, IL) following the manufacturers protocol. Samples were added to SDS-PAGE
buffer and boiled for 10 min. before loading onto a 4-12% Bis-Tris pre-cast gel (Invitrogen,
Carlsbad, CA) for electrophoresis. Proteins were transferred onto PVDF membranes (Bio
Rad, Hercules, CA) and blocked with AquaBlock™ (New England Biolabs, Ipswich, MA) for
1 hour at room temperature (RT). Primary antibody incubation was done in AquaBlock™
with rabbit anti-Met and anti-rabbit phospho-Met (Tyr1234/1235) (1:1000, Cell Signaling
Technology, Danvers, MA) overnight at 4°C. Alternating washes were done with PBS and
PBST. Secondary antibody (IRDye) (Rockland, Gilbertsville, PA) incubations were done in
AquaBlock™ for one hour at RT. Blots were imaged using LI-COR Odyssey Infrared
Imaging System (LI-COR Biosciences, Lincoln, NE).
Immunocytochemistry
Transfected neurons were treated, fixed and stained as previously described in
Chapter two. Briefly, cells were permeablized with 0.1% Triton X-100 detergent (Bio-Rad;
Hercules, CA) for 10 minutes. An 8% bovine serum albumin (Intergen Company;
Burlington, MA) in PBS was used to prevent non-specific binding for one hour at R.T.;
Primary antibody incubations were at a 1:2500 dilution (see below) in 1% BSA in PBS at 4°C
overnight. Secondary antibody, 1:3000 Alexafluor 488 goat-anti-mouse (Invitrogen:
Carlsbad, CA) was applied for two hours at room temperature. Coverslips were mounted
with ProLong Gold anti-fade reagent (Invitrogen; Carlsbad, CA) and all washes were done
with PBS. Imaging and analysis were performed as described above. For presynaptic
excitatory transmission the VGLUT1 (Synaptic Systems, Goettingen, Germany) marker
(Balschun, Moechars et al.) was employed and for general presynaptic transmission synapsin1
(Synaptic Systems, Goettingen, Germany) (Ferreira and Rapoport 2002) was applied. A
postsynaptic function was established by PSD-95 (Milipore, Billerica, MA) (El-Husseini,
Schnell et al. 2000). In each instance the total number of spines was counted for the
treatment groups, control, Nle1-AngIV and Dihexa, to ensure an active phenotype.
The total number of actin enriched spines (red) adjacent to VGLUT1 or Synapsin were
counted and converted to a percentage as the percent correlation of treatment-induced spines
to presynaptic markers is a strong indicator of ability to transmit excitatory signals. In our
application the number of correlations consisted of red fluorescent-tagged actin spines against
green PSD-95 immuno-positive puncta which, when merged, resulted in an orange spine.
Whole-cell recordings
Patch-clamp experiments were performed on mRFP-β-actin transfected cultured
hippocampal neurons (vehicle control) and on transfected hippocampal neurons with 1pM
Hinge or Dihexa, or 10 ng/ml HGF (R&D Systems) 5 day pretreatment. Recordings were
taken from neurons that were pyramidal-like in shape (~20 µm cell bodies and asymmetric
dendrite distribution). The time after transfection was 6 days. The culture medium was
exchanged by an extracellular solution containing (in mM) 140 NaCl, 2.5 KCl, 1 MgCl , 3
CaCl , 25 glucose, and 5 HEPES; pH was adjusted to 7.3 with KOH; osmolality was adjusted
to 310 mOsm. Cultures were allowed to equilibrate in a recording chamber mounted on
inverted microscope (IX-71; Olympus optical, Tokyo) for 30 min before recording.
Transfected cells were visualized with fluorescence (Olympus optical). Recording pipettes
were pulled (P-97 Flaming/Brown micropipette puller; Sutter Instrument, Novato, CA) from
standard-wall borosilicate glass without filament (OD = 1.5 mm; Sutter Instrument). The
pipette-to-bath DC resistance of patch electrodes ranged from 4.0 to 5.2MΩ, and were filled
with a internal solution of the following composition (in mM): 25 CsCl, 100 CsCH O S, 10
phosphocreatine, 0.4 EGTA, 10 HEPES, 2 MgCl , 0.4 Mg-ATP, and 0.04 Na-GTP; pH was
adjusted to 7.2 with CsOH; osmolality was adjusted to 296 - 300 mOsm. Miniature EPSCs
(mEPSCs) were isolated pharmacologically by blocking GABA receptor chloride channels
with picrotoxin (100 µM; Sigma), blocking glycine receptors with strychnine (1 µM; Sigma),
and blocking action potential generation with tetrodotoxin (TTX, 500 nM; Tocris).
Recordings were obtained using a Multiclamp 700B amplifier (Molecular Devices,
Sunnyvale, CA). Analog signals were low-pass Bessel filtered at 2 kHz, digitized at 10 kHz
through a Digidata 1440A interface (Molecular Devices), and stored in a computer using
Clampex 10.2 software (Molecular Devices). The membrane potential was held at -70 mV at
room temperature (25ºC) during a period of 0.5 – 2 h after removal of the culture from the
incubator. Liquid junction potentials were not corrected. Data analysis was performed using
Clampfit 10.2 software (Molecular Devices), and Mini-Analysis 6.0 software (Synaptosoft
Inc.; Fort Lee, NJ). The criteria for successful recording included the electrical resistance of
the seal between the outside surface of the recording pipette and the attached cell >2 GΩ,
neuron input resistance >240 MΩ. The mEPSCs had a 5-min recording time.
RESULTS
Hepatocyte Growth Factor Augments the dendritic architecture and Supports Synaptogenesis
Dihexa and Nle1-AngIV have previously been shown to induce spinogenesis in
mRFP-β-actin transfected hippocampal neurons (see Example 1); however the mechanism
underlying this action was unknown. Because of the ability of Norleual, another AngIV
analogue to block the action of HGF on c-Met (Yamamoto et al., 2010) we hypothesized that
increases in spine density initiated by Dihexa and Nle1-AngIV are mediated by the HGF/c-
Met system. As such, the effects of HGF on spinogenesis in dissociated hippocampal
cultures were evaluated. Hippocampal neurons were transfected with mRFP-β-actin on day
in vitro (DIV) 6 and stimulated with HGF for 5 days.
A dose-dependent increase in spine numbers following HGF stimulation was observed
with the lowest effective dose being 5 ng/ml dose (mean spine numbers = 24.7; **= p < 0.01
vs. control; ns vs HGF 10 and 20 ng/ml). The most significant effects were produced by 10
and 20ng/ml doses (mean spine numbers = 27.5 and 27.0 respectively; n = 50 per treatment
group; *** = p < 0.001; df = 4/245; F = 13.5). A 2.5 ng/ml dose of HGF, however, had no
effect on basal spine numbers (mean spine numbers = 18.6 vs. control = 18.0) (Figure 8) and
was therefore considered to be sub-threshold.
To evaluate the ability of HGF to augment spinogenesis in a more physiologically
relevant environment, organotypic hippocampal slices were employed. Hippocampal slices,
which were biolistically transfected with the soluble red fluorescent protein Tomato were
stimulated with 10 ng/ml HGF, 10 M Dihexa or vehicle for 48 hours. CA1 hippocampal
neurons, which are known to undergo plastic changes in response to learning were easily
singled out for analysis based on morphology. Dihexa and HGF significantly increased the
number of spines per 50 μm dendrite length in the CA1 hippocampal neurons (mean spine
numbers = 15.0 and 18.5 respectively compared to mean control spine numbers = 6.1; *** =
P < 0.001 and ** = P < 0.01 between treatment groups; df = 2/81; F = 41.5) (Figure 9A and
Previous studies in which neurons were treated with Dihexa and Nle1-AngIV
indicated that most of dendritic spines that were induced co-localized with both pre- and
postsynaptic markers indicated that these new spines supported functional synapses. In
addition, the majority of synaptic input appeared to be glutamatergic. Because Dihexa, Nle1-
AngIV, and HGF are proposed to all act through a common mechanism, the functional
properties of HGF-induced spines was evaluated. mRFP-β-actin transfected hippocampal
neurons were immunostained for a general marker of presynaptic active zones, synapsin
(Ferreira and Rapoport; 2002) as well as a marker specific to glutamatergic synapses,
Vesicular Glutamate Transporter 1 (VGLUT1) (Balschun, Moechars et al. 2010). HGF
stimulation significantly augmented the number of postsynaptic spines (mean number of
spines per 50 µm dendrite length for HGF = 33 vs. 23 for control; *** = P < 0.001; ± S.E.M.
by one-way ANOVA) thus ensuring an active phenotype by HGF-treatment (Figure 10A and
B). The number of postsynaptic spines adjacent to VGLUT1, or synapsin-positive puncta
were counted and converted to a percentage of the total spines counted. For HGF-treated
neurons (10 ng/ml) immunostained against Synapsin1 a 98% correlation between the
presynaptic marker and postsynaptic actin-enriched spine was observed (Figure 9C). A 95%
correlation for VGLUT1 and postsynaptic spines indicated that spines induced by HGF were
almost exclusively glutamatergic (Figure 10D). The correlation between green puncta and
red spines for vehicle treated neurons was similarly 94% for Synapsin and VGLUT1 (Figure
10C and D).
The above data suggest that spines produced in response to HGF-treatment form
functional synapses. Furthermore, the high correlation with VGLUT1 suggests that many of
these inputs are excitatory in nature. To further evaluate this conclusion, we measured the
frequency of spontaneous AMPA-mediated mini-excitatory postsynaptic currents (mEPSCs)
from neurons following HGF treatment and compared these data to those obtained for
Dihexa, which had previously established to increase mEPSC frequency. Recordings were
done on dissociated hippocampal neurons transfected with mRFP-β-actin and treated with 10
M Dihexa, 10 ng/ml HGF or an equivalent volume of vehicle for 5 days.
Both HGF (mean frequency = 7.09 ± 0.53; n = 11) and Dihexa treatment (mean frequency =
6.75 ± 0.99; n = 9) increased excitatory synaptic transmission nearly two-fold over control
(mean frequency = 3.55 ± 0.60; n = 9; ** = P < 0.002; mean ± S.E.M. by one-way ANOVA
followed by Newman-Keuls post hoc test) treated neurons (Figure 11), confirming the
supposition that HGF treatment supports increased synaptogenesis.
In order to ascertain whether angiotensin IV ligand actions are mediated by HGF/c-
Met a synergy experiment was performed. Sub-threshold doses of HGF augmented with sub-
threshold doses of Dihexa or Nle1-AngIV were previously shown to promote spinogenesis,
suggesting a common mechanism of action. Dissociated hippocampal neurons transfected
with mRFP-β-actin were stimulated for 5 days with sub-threshold concentrations of HGF and
Dihexa (2.5 ng/ml + 10 M, respectively), biologically active doses of HGF (10 ng/ml),
Dihexa or Nle1-AngIV (10 M) or a combination of sub-threshold doses of 2.5 ng/ml HGF
-12 -12
+ 10 M Dihexa or 2.5 ng/ml HGF + 10 M Nle1-AngIV. The results are presented in
Figures 12 A and B. Sub-threshold concentrations of HGF (2.5 ng/ml), Dihexa and Nle1-
AngIV (10 M) had no effect on basal spinogenesis and did not differ from control treated
neurons (mean ± S.E.M. spine numbers for control = 17.4, HGF = 16.5, Dihexa = 17.1 and
Nle1-AngIV = 16.5 per 50 μm dendrite length; p > 0.05). Biologically active doses of HGF
(10 ng/ml), Dihexa and Nle1-AngIV (10 M) produced a significant effect over control
treated spines (mean ± S.E.M. spine numbers for HGF = 29.3, Dihexa = 26.4 and Nle1-
AngIV = 29.8 per 50 µm dendrite). Combined sub-threshold doses of 2.5 ng/ml + 10 M
Dihexa and 2.5 ng/ml + 10 M Nle1-AngIV phenocopied the effects of each agonist at its
biologically active dose alone (mean ± S.E.M. spine numbers for HGF + Dihexa are 28.8 and
HGF + Nle1-AngIV are 26.2 per 50 μm dendrite length compared to control treated neurons =
17.4; *** = P < 0.001; mean ± S.E.M.; by one-way ANOVA followed by Tukey post hoc
test).
Seeking further substantiation for angiotensin IV ligand and HGF/c-Met mediated
interactions, the novel HGF antagonist Hinge (DYIRNC, SEQ ID NO: 3) was utilized (Kawas
et al., 20113 Hinge was confirmed as an HGF/c-Met receptor antagonist by its ability to
inhibit scattering of Madin-Darby canine kidney (MDCK) cells, the gold standard for
assessment of c-Met mediated activity. Cell scattering involves a loss of cell adhesion
properties, cell migration and differentiation, the hallmarks of HGF and c-Met actions
(Yamamoto, Elias et al., 2010; Birchmeier, Sonnenberg et al. 1993). Hinge was tested for its
effects on dissociated hippocampal neurons and was found to have no effect on spinogenesis
over a wide range of doses, thus indicating that Hinge and the HGF/c-Met system do not have
a significant role in the basal spinogenesis seen in the cultured neurons (Figure 13A).
However, Hinge did effectively inhibit spine formation in neurons stimulated with 10 ng/ml
-12 -12
HGF (Figure 13B), 10 M Nle1-AngIV (Figure 13C) or 10 M Dihexa (Figure 12D)
further supporting the contention that these actions are mediated by the HGF/c-Met system.
To assess the effects of Hinge on excitatory synaptic transmission mEPSCs were
recorded form mRFP-β-actin transfected hippocampal neurons treated for 5 days with Hinge
-12 -12 -12
(10 M), HGF (10 ng/ml), Dihexa (10 M), Hinge + HGF (10 M + 10 ng/ml,
respectively) or Hinge + Dihexa (10 M each). Hinge alone does not affect synaptic
transmission (mean frequency = 4.51 ± 0.47) compared to vehicle treated neurons (mean
frequency = 5.31 ± 0.35; Figure 14A and B). HGF and Dihexa frequencies were
significantly increased compared to both Hinge and vehicle treated neurons (mean frequency
for HGF = 9.66 ± 0.20 and for Dihexa = 8.25 ± 0.56). However these effects are significantly
attenuated by stimulation in the presence of Hinge (mean frequencies for HGF + Hinge = 5.25
± 0.27 and Dihexa + Hinge = 5.57 ± 0.65; Figure 14A and B). These results suggest that the
newly generated spines are forming functional synapses and while Hinge has no effect on
synaptic transmission, it is its ability to inhibit spinogenesis that attenuates the AMPA-
mediated frequencies.
The proposed angiotensin IV receptor HGF is the ligand for the tyrosine kinase
receptor c-Met. Although the localization of c-Met and HGF mRNA in the brain has been
well documented (Jung, Castren et al. 1994; Honda, Kagoshima et al. 1995; Thewke and
Seeds 1996; Achim, Katyal et al. 1997) the presence and distribution of c-Met protein has not
been examined. Therefore we probed several brain regions for the presence of c-Met but
were unable to do so for HGF due to a lack of effective antibodies. High levels of c-Met
protein were observed throughout most of the brain regions. Specifically, the highest signal
of c-Met protein was seen in the hippocampus and appears to be greater than in the liver
which is a major site of HGF production. A strong signal was also observed in the prefrontal
cortex and midbrain, regions of importance to cognition, while neocortex had a somewhat
attenuated signal the cerebellum produced the lowest signal (Figures 15 A and B).
The apparent dependency of the actions of Dihexa on the HGF/c-Met system
predicted that Dihexa in the presence of sub-threshold levels of HGF should be able to
stimulate c-Met phosphorylation and activation. Therefore acute adult rat hippocampal slices
were stimulated with HGF, Dihexa at saturating and non-saturating concentrations alone and
in combination and probed for phospho-Met. Phosphorylation of the c-Met receptor indicates
receptor activation. Figure 16 shows phosphorylation of the c-Met receptor following a 30
minute treatment with vehicle and various concentrations HGF or Dihexa. Saturating doses
of HGF (100 ng/ml) and Dihexa (10 M) Dihexa both increased c-Met phosphorylation
compared to control (aCSF) treated slices; (p < 0.007). Non-saturating doses of HGF (50
ng/ml) and Dihexa (10 M) were not statistically different from control treated slices (p >
0.05) and therefore considered to be sub-threshold. The sub-threshold doses of HGF and
Dihexa combined, however, appeared to produce an effect similar to the saturating doses of
HGF and Dihexa (p < 0.007). Thus dependent on the dose it appears that Dihexa is
independently capable of activating the HGF/c-Met system in the adult rat brain alone as well
as in conjunction with HGF. In concert with these findings Dihexa able to dramatically
augment the ability of HGF to activate c-Met by phosphorylation in HEK293 cells (Figure
17) and stimulate MDCK cell scattering (Figure 18).
To irrefutably confirm that the AngIV analogues act via the HGF/c-met system an
shRNA for c-Met was employed to knock-down the receptor. Dissociated hippocampal
neurons were transfected with mRFP-β-actin and shMet RNA and receptor knock-down was
allowed to take place for 48 hours prior to stimulating with 0.5 µg (per well) HGF (10 ng/ml),
Dihexa or Nle1-AngIV (both at 10 M). Longer exposure appeared to be detrimental or
toxic to the neurons. Effective c-Met receptor knock-down was verified by transfecting
human embryonic kidney (HEK) cells with (0.1 µg) 6-Myc- tagged c-Met, (0.1 µg)shMet or
mRFP-β-actin alone. Successful knockdown was confirmed by immunoblotting for Myc
tagged c-met using an anti-Myc antibody (Figure 19).
Neurons transfected with mRFP-β-actin alone, serving as the control, were treated
with 10 ng/ml HGF, 10 M Dihexa or Nle1-AngIV. A significant increase in the number of
spines compared to control treated neurons was observed (mean spine numbers per 50 µm
dendrite length = 13.2 vs HGF = 20.6; Dihexa = 21.8 and Nle1-AngIV = 20.0; p < 0.05 by
one-way ANOVA followed by Tukey post hoc test). Neurons transfected with mRFP-β-actin
and shMet that were stimulated with 10 ng/ml HGF, 10 M Dihexa or Nle1-AngIV, did not
differ from control in terms of spine numbers (mean spine numbers per 50 µm dendrite length
= 13.5 vs HGF = 12.4; Dihexa = 12.0 and Nle1-AngIV = 12.1; p > 0.05 by one-way ANOVA
followed by Tukey post hoc test) as shown in Figure 20. A scrambled RNA sequence was
employed as the negative control and had no effect on basal or stimulated spinogenesis (data
not shown). These results confirm that the effects of AngIV analogs are mediated by the
HGF/c-Met system.
The Morris water maze, a hippocampal-dependent spatial learning task requiring rats
to locate a pedestal hidden beneath the surface of the water by orienting themselves to extra-
maze cues was employed to evaluate the impact of the HGF antagonist, Hinge, on the pro-
cognitive effects of Dihexa. The groups tested included aCSF followed by aCSF,
scopolamine (70 nM) followed by aCSF, scopolamine followed by Dihexa (300 pM), aCSF
followed by Hinge (300 pM) and scopolamine + Hinge followed by Dihexa. Figure 21
represents the mean latencies to find the hidden pedestal for days 1-8 of training in the water
maze. None of the groups differed significantly in latency to find the pedestal on day one of
training. Mean latencies for the vehicle control (aCSF → aCSF) group = 89.3 s; the
scopolamine treated group = 114.7 s; the scopolamine + Hinge → Dihexa treated group
latency = 107.9 s; the Hinge group mean latency = 111.1 s; and the scopolamine → Dihexa
group = 115.2 s. By the fourth day of training, considered to be a crucial day on which the
most improvement in training and neural plasticity occurs (Meighan et al., 2006), the
scopolamine group (mean latency to find the pedestal = 102.4 s) and the scopolamine + Hinge
→ Dihexa group (mean latency = 105.2 s) showed no signs of improvement compared to the
vehicle control group (mean latency = 43.0 s), the Hinge group (mean latency = 78.3 s) and
the scopolamine →Dihexa group (mean latency = 63.0 s). On the final day of training when
maximal learning has occurred (Meighan, Meighan et al. 2006) the mean latencies for the
scopolamine group (mean latency to find the pedestal = 84.8 s) and the scopolamine + Hinge
→ Dihexa group (mean latency = 93.6 s) indicated little improvement in learning compared
to the vehicle control group (mean latency = 43.0 s), the Hinge group (mean latency = 46.1 s)
and the scopolamine → Dihexa group (mean latency 62.3 s). These results suggest that HGF
and c-Met play an important role in hippocampal-dependent cognitive processes.
Discussion
The pro-cognitive effects of angiotensin IV analogues suggest that anti-dementia
drugs based on this system can be developed (Braszko, Kupryszewski et al. 1988; Stubley-
Weatherly, Harding et al. 1996; Pederson, Harding et al. 1998; Wright, Stubley et al. 1999).
However, due to poor metabolic stability of angiotensin IV and many AngIV analogues, the
inability of early analogues to penetrate the blood brain barrier, and the failure to identify the
AT4 receptor, no pharmaceutical company has moved forward with their development.
Dihexa, a novel angiotensin IV analogue synthesized by our laboratory, is stable and orally
active and has thus overcome the major pharmacokinetic impediments preventing
development. Dihexa has been proven to be stable in the blood for over 5 hours (not shown),
survived passage through the gut to penetrate the blood brain barrier, and overcomes
cognitive deficits in acute and chronic models of dementia (not shown). A general
mechanism, established for facilitation of the water maze task, involves expansion of the
dendritic arbor in the form of newly developed postsynaptic spines and accompanying
synaptogenesis. The last remaining hurdle to development was the lack of a molecular
mechanism.
Here we demonstrate that the actions of AngIV analogues are dependent on the
HGF/c-Met system. Both systems appear to mediate similar physiological effects. The
Angiotensin IV/AT4 system has cerebroprotective effects (Wright, Clemens et al. 1996; Date,
Takagi et al. 2004), augments long term potentiation (Kramar, Armstrong et al. 2001;
Wayner, Armstrong et al. 2001; Akimoto, Baba et al. 2004; Davis, Kramar et al. 2006), has
well established pro-cognitive effects (Wright and Harding 2008), and is suspected to regulate
neural stem cell development. The HGF/c-Met system also has pro-cognitive effects
(Akimoto, Baba et al. 2004; Tyndall and Walikonis 2006; Tyndall and Walikonis 2007) and
is known to be involved in stem cell regulation (Urbanek, Rota et al. 2005; Nicoleau,
Benzakour et al. 2009). In addition to functional similarities there is sequence homology
between angiotensin IV and the “hinge” linker region of HGF (Wright, Yamamoto et al.
2008). This notion was further solidified by the observation that the well known AT4
antagonist, Norleual, is capable of blocking many HGF/c-Met regulated functions such as
MDCK cell scattering (Yamamoto, Elias et al.2010).
Facilitation of the water maze task is effected by Dihexa and the parent angiotensin IV
ligand, Nle1-AngIV, by augmentation of neurotransmission occurring through elaboration of
the dendritic arbor. The hypothesized linkage between the action of AngIV analogues and the
HGF/c-Met system predicted that like Dihexa and Nle1-AngIV HGF should be able to
stimulate dendritic spine growth in dissociated hippocampal neurons.
As predicted, HGF promoted a dose-dependent increase in spinogenesis (Figure 7) in
dissociated hippocampal neurons. The most effective concentration of HGF (10 ng/ml) was
subsequently found to stimulate hippocampal neurons in organotypic hippocampal slice
cultures which are more intact preparations similar to Dihexa (Figure 8A and B) further
establishing a mechanistic link between Dihexa and HGF/c-Met. To evaluate the
physiological relevance of these new spines and to determine the neurotransmitter signature
of resident synapses, HGF treatment-induced spines labeled with mRFP-β-actin were
immunostained for the universal presynaptic marker Synapsin that is located in the
presynaptic active zones (Ferreira and Rapoport 2002) and the excitatory presynaptic marker
VGLUT1 that is found at glutamatergic presynaptic synapses (Balschun, Moechars et al.).
The ratio of postsynaptic mRFP-β-actin labeled spines juxtaposed to Synapsin or VGLUT1
spines was not different from control treated neurons suggesting treatment-induced spines are
forming functional synapses (Figure 9A-D). Further validation of synaptogenesis was
obtained by recording mEPSCs, spontaneous presynaptic bursts independent of action
potentials, on HGF and Dihexa treated neurons. AMPA-mediated transmission was
amplified in response to HGF and Dihexa treatment as shown by increased frequencies
(Figure 10).
Sub-threshold concentrations of Dihexa and HGF or Nle1-AngIV and HGF were used
to stimulate hippocampal neurons in vitro to determine whether the angiotensin IV ligands
Dihexa and Nle1-AngIV, and HGF affect the same signaling cascade or act on one receptor
(c-Met). To determine whether Dihexa and Nle1-AngIV engage the same signaling cascade
sub-threshold concentrations of AngIV ligands were combined with sub-threshold doses of
HGF. While sub-threshold concentrations of each ligand alone did not alter basal
spinogenesis, combined sub-threshold concentrations of 10 M Dihexa and 2.5 ng/ml HGF
or 10 M Nle1-AngIV and 2.5 ng/ml of HGF produced a near ceiling effect, similar to
biological responsive doses of each ligand alone (Figure 11A and B). The similarities in the
dendritic responses to the AngIV analogues and HGF are consistent with a common
mechanism of action.
To further strengthen this perceived commonality of mechanism, the novel HGF
antagonist Hinge was employed and evaluated for its effects on hippocampal neurons
stimulated with AngIV analogues and HGF. Hinge, like the angiotensin IV antagonist
Norleual, was established as a c-Met antagonist by its ability to block HGF-dependent c-Met
phosphorylation and prevent HGF-dependent scattering in the MDCK epithelial cell line.
Cell scattering, which is the hallmark of an HGF/c-Met interaction, leads to a loss of cell
adhesion properties that allow cells to migrate (Yamamoto, Elias et al.; Birchmeier,
Sonnenberg et al. 1993). Hinge was found to have no adverse effects on cultured
hippocampal neurons and did not promote or hinder spinogenesis (Figure 12A). At pico
molar concentrations, however, Hinge prevented HGF, Nle1-AngIV and Dihexa induced
spinogenesis (Figure 12B-D) further suggesting that the effects observed for our angiotensin
IV ligands are HGF/c-Met mediated. The effects of Hinge on synaptogenesis were evaluated
by recording mEPSC frequencies on cultured hippocampal neurons. While Hinge alone did
alter base-line synaptic transmission it attenuated HGF and Dihexa increases in AMPA-
frequencies (Figure 13 A and B). This effect was likely due to attenuation of spinogenesis
promoted by HGF and Dihexa treatments since, without the antagonizing effect of Hinge,
each agonist increased mini-AMPA frequencies (Figure 13 A-B and Figure 10) thus forming
functional synaptic connections. Taken together, these data suggest that inhibiting HGF does
not alter the number of functional synapses in vehicle treated neurons but attenuates the
effects of HGF and Dihexa on synaptogenesis by decreasing the number of postsynaptic
spines.
To additionally support the contention that the agonists Dihexa and Nle1-AngIV are
acting through HGF and its receptor c-Met, hippocampal neurons were transfected with
shRNA to knockdown the c-Met receptor. Knockdown of the receptor was verified by
immunoblotting against a Myc-tagged c-Met gene product (Figure 16). As expected,
stimulation of hippocampal neurons transfected with mRFP-β-actin with HGF, Dihexa and
Nle1-AngIV had significantly enhanced dendritic arbors while those additionally transfected
with shc-Met RNA were no different from control treated neurons (Figure 17). These data
provide conclusive support for our belief that angiotensin IV ligands Dihexa and Nle1-AngIV
act through the HGF/c-Met system.
The newly developed angiotensin IV agonist ligand Dihexa has been shown to
facilitate acquisition of a spatial learning and memory task in scopolamine treated rats (data
not shown). Because it is prohibitively expensive to test HGF in the water maze, we instead
evaluated its involvement in cognition by employing the HGF antagonist Hinge to block the
actions of Dihexa. Treatment with the muscarinic cholinergic receptor antagonist
scopolamine renders rats acutely amnesic and therefore unable to learn the task. A rescue
effect is observed in rats that are given Dihexa following scopolamine pretreatment. These
rats exhibit rapid facilitation of the task and did not perform differently from vehicle treated
rats. The group of rats that was pretreated with a scopolamine and Hinge did not display the
rescue effect observed by Dihexa in the scopolamine preparation (Figures 14A and B). These
data demonstrate a function for HGF and c-Met system in learning and memory, and that
agents which mimic the action of HGF can be used to enhance learning and memory in
subjects in need thereof.
EXAMPLE 3: Development of Antiotensin IV Analogs as Hepatocyte Growth
Factor/Met Modifiers
The 6-AH family [D-Nle-X-Ile-NH-(CH ) -CONH ; where X= various amino acids]
2 5 2
of Angiotensin IV analogs, bind directly to Hepatocyte Growth Factor (HGF) and inhibit
HGF’s ability to form functional dimers. The metabolically stabilized 6-AH family member,
D-Nle-Tyr-Ile-NH-(CH ) -CONH , had a t in blood of 80 min compared to the parent
2 5 2 1/2
compound Norleual (Nle-Tyr-Leu-Ψ-(CH -NH ) -His-Pro-Phe, SEQ ID NO: 1), which had
a t in blood of < 5 min. 6-AH family members were found to act as mimics of the
dimerization domain of HGF (hinge region), and inhibited the interaction of an HGF
molecule with a H-hinge region peptide resulting in an attenuated capacity of HGF to
activate its receptor Met. This interference translated into inhibition of HGF-dependent
signaling, proliferation, and scattering in multiple cell types at concentrations down into the
low picomolar range. We also noted a significant correlation between the ability of the 6-AH
family members to block HGF dimerization and inhibition of the cellular activity. Further, a
member of the 6-AH family with cysteine at position 2, was a particularly effective antagonist
of HGF-dependent cellular activities. This compound suppressed pulmonary colonization by
B16-F10 murine melanoma cells, which are characterized by an overactive HGF/Met system.
Together these data indicate that the 6-AH family of AngIV analogs exert their biological
activity by modifying the activity of the HGF/Met system and offer the potential as
therapeutic agents in disorders that are dependent on or possess an over-activation of the
HGF/Met system.
INTRODUCTION
The multifunctional growth factor hepatocyte growth factor (HGF) and its receptor
Met are important mediators for mitogenesis, motogenesis, and morphogenesis in a wide
range of cell types (Birchmeier et al., 2003) including epithelial (Kakazu et al., 2004),
endothelial (Kanda et al., 2006), and hematopoietic cells (Ratajczak et al., 1997), neurons
(Thompson et al., 2004), melanocytes (Halaban et al., 1992), and hepatocytes (Borowiak et
al., 2004). Furthermore, dysregulation of the HGF/Met system often leads to neoplastic
changes and to cancer (in both human and animal) where it contributes to tumor formation,
tumor metastasis, and tumor angiogenesis (Christensen et al., 2005; Liu et al., 2008). Over-
activation of this signaling system is routinely linked to poor patient prognosis (Liu et al.,
2010). Therefore molecules that inhibit the HGF/Met system can be expected to exhibit anti-
cancer activity and attenuate malignant and metastatic transformations.
HGF is a vertebrate heteromeric polypeptide growth factor with a domain structure
that closely resembles the proteinases of the plasminogen family (Donate et al., 1994). HGF
consists of seven domains: an amino terminal domain, a dimerization-linker domain, four
kringle domains (K1-K4), and a serine proteinase homology (SPH) domain (Lokker et al.,
1992; Chirgadze et al., 1999). The single chain pro-polypeptide is proteolytically processed
by convertases to yield a mature α (heavy chain 55 KDa), and β (light chain 34 KDa)
heterodimer, which are bound together by a disulfide link (Stella and Comoglio, 1999;
Birchmeier et al., 2003; Gherardi et al., 2006). In addition to proteolytic processing, HGF
requires dimerization to be fully activated (Lokker et al., 1992; Chirgadze et al., 1999; Youles
et al., 2008). Several reports have shown that HGF forms dimers and/or multimers, which are
arranged in a head-to-tail orientation, prior to its interaction with Met (Gherardi et al., 2006).
The dimer interface, which encompasses the inter-domain linker amino acids (K122, D123,
Y124, I125, R126, and N127) is referred to as the hinge region (Gherardi et al., 2006; Youles
et al., 2008). Although both pre-pro-HGF and the active disulfide-linked heterodimer bind
Met with high affinity, it is only the heterodimer that is capable of activating Met (Lokker et
al., 1992; Sheth et al., 2008).
Recent studies from our laboratory (Yamamoto et al., 2010) have shown that
picomolar concentrations of the AngIV analog, Norleual (Nle-Tyr-Leu-ψ-(CH -NH ) -His-
Pro-Phe), are capable of potently inhibiting the HGF/Met system and bind directly to the
hinge region of HGF blocking its dimerization (Kawas et al., 2011). Moreover, a hexapeptide
representing the actual hinge region possessed biochemical and pharmacological properties
identical to Norleual’s (Kawas et al., 2011). The major implication of those studies was that
molecules, which target the dimerization domain of HGF, could represent novel and viable
anti-cancer therapeutics. Additionally, these data support the development of such molecules
using Norleual and/or the Hinge peptide as synthetic templates.
Despite its marked anti-cancer profile Norleual is highly unstable making its transition
to clinical use problematic. Thus a family of metabolically stabile Ang IV-related analogs has
been developed in our laboratory, which are referred to here as the 6-AH family because of 6-
amnio hexanoic amide substituted at the C-terminal position. This substitution along with D-
norleucine at the N-terminal enhances the metabolic resistance of family members.
In this Example 3, it is demonstrated that 6-AH family members (i.e., HGF Mimics)
have superior metabolic stability when compared to Norleual, bind to HGF with high affinity,
and act as hinge region mimics; thus preventing HGF dimerization and activation. This
interference translates into inhibition of HGF-dependent signaling, proliferation, and
scattering in multiple cell types at concentration in the picomolar range. A positive
correlation was evident between the ability to block dimerization and the inhibition of the
cellular outcomes of HGF activation. Finally D-Nle-Cys-Ile-NH-(CH ) -CONH , a member
2 5 2
of the 6-AH family suppressed pulmonary colonization by B16-F10 murine melanoma cells,
which are characterized by an overactive HGF/Met system. This Example highlights the
ability of AngIV-like molecules to bind to HGF, block HGF dimerization, and inhibit the
HGF/Met system. Moreover, these HGF mimics have utility as AngIV-related
pharmaceuticals and can function as therapeutic agents in disorders where inhibition of the
HGF/Met system would be clinically advantageous.
MATERIAL AND METHODS
Animals. C57BL/6 mice from Taconic farms were used in the lung colonization
studies. Male Sprague-Dawley rats (250+ g) were obtained from Harlan Laboratories (CA,
USA) for use in pharmacokinetic studies. Animals were housed and cared for in accordance
with NIH guidelines as described in the “Guide for the Care and Use of Laboratory Animals”.
Compounds. D-Nle-X-Ile-NH-(CH ) -COOH; where X= various amino acids and
Norleual (Nle-Tyr-Leu-ψ-(CH -NH ) -His-Pro-Phe, SEQ ID NO: 1) were synthesized using
Fmoc based solid phase methods in the Harding laboratory and purified by reverse phase
HPLC. Purity and structure were verified by LC-MS. Hepatocyte growth factor (HGF) was
purchased from R&D Systems (Minneapolis, MN).
Antibodies. Anti-Met was purchased from Cell Signaling Technology (Beverly, MA)
and the phospho-Met antibody was purchased from AbCam, Inc (Cambridge,MA).
Cell culture. Human embryonic kidney cells 293 (HEK293) and Madin Darby canine
kidney cells (MDCK) were grown in DMEM, 10% fetal bovine serum (FBS). Cells were
grown to 100% confluency before use. HEK and MDCK cells were serum starved for 2-24 h
prior to the initiation of drug treatment.
Blood Stability Studies. To compare the blood stability of Norleual and D-Nle-Tyr-
Ile-NH-(CH ) -CONH , a representative member of the 6-AH family, 20 µL of compound-
2 5 2
containing vehicle (water [Norleual] or 30% ethanol [D-Nle-Tyr-Ile-NH-(CH ) -CONH ])
2 5 2
was added to 180 µL of heparinized blood and incubated at 37 C for various times. For
Norleual, 37 C incubations were stopped at 0, 20, 40, and 60 min, and for D-Nle-Tyr-Ile-NH-
(CH ) -CONH , incubations were stopped at 0, 1, 3 and 5 h.
2 5 2
At the end of each incubation, 20 µL of Nle - AngIV (100 µg/ mL) was added to each
sample as an internal standard. D-Nle-Tyr-Ile-NH-(CH ) -CONH samples were centrifuged
2 5 2
at 4 C for 5 min at 2300x g to pellet erythrocytes, and the plasma was transferred to clean
tubes. The Norleual and D-Nle-Tyr-Ile-NH-(CH ) -CONH samples were precipitated by
2 5 2
adding 3 vol of ice-cold acetonitrile (ACN) and the samples were vortexed vigorously. All
samples were centrifuged at 4 C, 2300x g for 5 min and the supernatants were transferred to
clean tubes. Samples were then evaporated to dryness in a Savant SpeedVac® concentrator
(Thermo Fisher Scientific, Waltham, MA) , the residue was reconstituted in 225 µl 35%
methanol, vortexed briefly, transferred to HPLC autosampler vials, and 100 µl injected into
the HPLC system.
Samples were then separated by HPLC on an Econosphere C18 (100mm x 2.1mm)
from Grace Davison Discovery Science (Deerfield, IL). Peaks were detected and analyzed by
mass spectrographic methods using a LCMS-2010EV mass spectrometer (Shimadzu, Kyoto
Japan). The mobile phase consisted of HPLC water (Sigma St. Louis, MO) with 0.1%
trifluoroacetic or 0.1% heptafluorobutyric acid (Sigma St. Louis, MO) and varying
concentrations of ACN or methanol. Separation was carried out using a gradient method, at
ambient temperature and a flow rate of 0.3 mL/min (see below for more information).
Stability half-lives were determined assuming a normal single phase exponential decay using
Prism 5 graphical/statistical program (GraphPad, San Diego, CA).
IV Pharmacokinetics.
Surgerical Procedures. Male Sprague-Dawley rats (250+ g) were allowed food (Harlan
Teklad rodent diet) and water ad libitum in our AAALAC certified animal facility. Rats were
housed in temperature-controlled rooms with a 12 h light/dark cycle. The right jugular veins
of the rats were catheterized with sterile polyurethane Hydrocoat catheters (Access
Technologies, Skokie, IL, USA) under ketamine (Fort Dodge Animal Health, Fort Dodge, IA,
USA) and isoflurane (Vet One , MWI, Meridian, ID, USA) anesthesia. The catheters were
exteriorized through the dorsal skin. The catheters were flushed with heparinized saline
before and after blood sample collection and filled with heparin-glycerol locking solution (6
mL glycerol, 3 mL saline, 0.5 mL gentamycin (100mg/mL), 0.5 mL heparin (10,000 u/mL))
when not used for more than 8 h. The animals were allowed to recover from surgery for
several days before use in any experiment, and were fasted overnight prior to the
pharmacokinetic experiment.
Pharmacokinetic Study. Catheterized rats were placed in metabolic cages prior to the
start of the study and time zero blood samples were collected. Animals were then dosed
intravenously via the jugular vein catheters, with D-Nle-Tyr-Ile-NH-(CH ) -CONH
2 5 2
(24mg/kg) in 30% ethanol. After dosing, blood samples were collected as follows (times and
blood volumes collected are listed in chronological order):
Compound Time (min) Blood Volume Collected (µl)
D-Nle-Tyr-Ile- 0, 12, 30, 60, 90, 120, 180, 200, 200, 200, 200, 200, 300, 400, 500,
NH-(CH ) - 240, 300 500
CONH
After each blood sample was taken, the catheter was flushed with saline solution and a
volume of saline equal to the volume of blood taken was injected (to maintain total blood
volume).
Blood Sample Preparation. Upon collection into polypropylene microfuge tubes
without heparin, blood samples were immediately centrifuged at 4 C, 2300x g for 5 min to
remove any cells and clots and the serum transferred into clean microcentrifuge tubes. A
volume of internal standard (Nle -AngIV, 100 µg/mL) equal to 0.1 times the sample serum
volume was added. A volume of ice-cold acetonitrile equal to four times the sample serum
volume was then added and the sample vortexed vigorously for 30 s. The supernatants were
transferred to clean tubes, then held on ice until the end of the experiment, and stored at 4ºC
afterward until further processing.
Serial dilutions of D-Nle-Tyr-Ile-NH-(CH ) -CONH in 30% ethanol were prepared
2 5 2
from the stock used to dose the animals for standard curves. 20 µL of each serial dilution was
added to 180 µL of blood on ice for final concentrations of 0.01µg/mL, 0.1µg/mL, 1µg/mL
and 10µg/mL. The samples were centrifuged at 4 C, 2300x g for 5 min and the serum
transferred into polypropylene microcentrifuge tubes. A volume of internal standard (Nle -
AngIV, 100µg/mL) equal to 0.1 times the sample serum volume was added. A volume of ice-
cold acetonitrile equal to four times the sample serum volume was then added and the sample
vortexed vigorously for 30 s. The supernatants were transferred to clean tubes and samples
stored at 4ºC and processed alongside the pharmacokinetic study samples. All samples were
evaporated to dryness in a Savant SpeedVac® concentrator. The residue was reconstituted in
225 µl 35% methanol and vortexed briefly. The samples were then transferred to HPLC
autosampler vials and 100 µl was injected into the HPLC system a total of 2 times (2
HPLC/MS analyses) for each sample.
Chromatographic System and Conditions. The HPLC/MS system used was from
Shimadzu (Kyoto, Japan), consisting of a CBM-20A communications bus module, LC-20AD
pumps, SIL-20AC auto sampler, SPD-M20A diode array detector and LCMS-2010EV mass
spectrometer. Data collection and integration were achieved using Shimadzu LCMS solution
software. The analytical column used was an Econosphere C18 (100mm x 2.1mm) from
Grace Davison Discovery Science (Deerfield, IL, USA). The mobile phase consisted of
HPLC grade methanol and water with 0.1% trifluoroacetic acid. Separation was carried out
using a non-isocratic method ( 40% - 50% methanol over 10 min) at ambient temperature and
a flow rate of 0.3 mL/min. For MS analysis, a positive ion mode (Scan) was used to monitor
the m/z of D-Nle-Tyr-Ile-NH-(CH ) -CONH at 542 and the m/z of Nle -AngIV (used for
2 5 2
internal standard) at 395. Good separation of D-Nle-Tyr-Ile-NH-(CH ) -CONH and the
2 5 2
internal standard in blood was successfully achieved. No interfering peaks co-eluted with the
analyte or internal standard. Peak purity analysis revealed a peak purity index for D-Nle-Tyr-
Ile-NH-(CH ) -CONH of 0.95 and the internal standard of 0.94. D-Nle-Tyr-Ile-NH-(CH ) -
2 5 2 2 5
CONH eluted at 5.06 min and the internal standard at 4.31 min. Data were normalized based
on the recovery of the internal standard.
Pharmacokinetic Analysis. Pharmacokinetic analysis was performed using data from
individual rats. The mean and standard deviation (SD) were calculated for the group. Non-
compartmental pharmacokinetic parameters were calculated from serum drug concentration-
time profiles by use of WinNonlin® software (Pharsight, Mountain View, CA, USA). The
following relevant parameters were determined where possible: area under the concentration-
time curve from time zero to the last time point (AUC ) or extrapolated to infinity (AUC
0-last 0-
), C concentration in plasma extrapolated to time zero (C ), terminal elimination half-life
∞ max 0
(t ), volume of distribution (Vd), and clearance (CL).
Microsomal Metabolism. Male rat liver microsomes were obtained from Celsis
(Baltimore, MD, USA). The protocol from Celsis for assessing microsomal- dependent drug
metabolism was followed with minor adaptations. An NADPH regenerating system (NRS)
was prepared as follows: 1.7 mg/mL NADP, 7.8 mg/mL glucosephosphate and 6 units/mL
glucosephosphate dehydrogenase were added to 10 mL 2% sodium bicarbonate and used
immediately. 500 µM solutions of Norleual, D-Nle-Tyr-Ile-NH-(CH ) -CONH , piroxicam,
2 5 2
verapamil and 7-ethoxycoumarin (low, moderate and highly metabolized controls,
respectively) were prepared in acetonitrile. Microsomes were suspended in 0.1M Tris buffer
(pH 7.38) at 0.5 mg/mL and 100 µL of the microsomal suspension was added to pre-chilled
microcentrifuge tubes on ice. To each sample, 640 µL 0.1M Tris buffer, 10 µL 500 µM test
compound, and 250 µL of NRS was added. Samples were incubated in a rotisserie
hybridization oven at 37ºC for the appropriate incubation times (10, 20, 30 40 or 60 min).
500 µL from each sample was transferred to tubes containing 500 µL ice-cold acetonitrile
with internal standard per incubation sample. Standard curve samples were prepared in
incubation buffer and 500 µL added to 500 µL ice-cold acetonitrile with internal standard. All
samples were then analyzed by high performance liquid chromatography/mass spectrometry.
Drug concentrations were determined and loss of parent relative to negative control samples
containing no microsomes was calculated. Clearance was determined by nonlinear regression
analysis for k and t and the equation Cl = k Vd. For in vitro-in vivo correlation, Cl per
e 1/2 int e int
kg body weight was calculated using the following measurements for Sprague-Dawley rats:
44.8 mg of protein per g of liver, 40 g of liver per kg of body weight.
HGF Binding. The binding of 6-AH analogs to HGF was assessed by competition
using a soluble binding assay. 250µl of PBS containing human HGF (1.25ng) were incubated
with H-Hinge, the central dimerization domain of HGF, in the presence of varying
-13 -7
concentrations of 6-AH analogs between 10 M to 10 M (half-log dilutions) for 40 min at
37 C. The incubates were then spun through Bio-Gel P6 spin columns (400 μl packed
volume) for 1 min to separate free and bound H-Hinge and the eluent was collected. Five
milliliters of scintillation fluid was added to the eluent, which contained the HGF bound H-
Hinge, and was then counted using scintillation counter. Total disintegrations per minute of
bound H-Hinge were calculated based on machine counting efficiency. The Ki values for the
binding of the peptides were determined using the Prism 5. Competition binding curves were
performed in triplicate. Preliminary kinetic studies indicated that equilibrium binding was
reached by 40 min of incubation at 37 C. H- Hinge has recently been shown to bind to HGF
with high affinity (Kawas et al., 2011).
HGF Dimerization. HGF dimerization was assessed using PAGE followed by silver
staining (Kawas et al., 2011). Human HGF at a concentration of 0.08ng/μl with or without 6-
AH analogs was incubated with heparin at a final concentration of 5μg/ml. Loading buffer
was then added to each sample and the mixture separated by native PAGE using gradient
Criterion XT precast gels (4-12% Bis-Tris; Biorad Laboratories, Hercules, CA). Next the gel
was silver stained for the detection of the HGF monomers and dimers. Bands were
quantitated from digital images using a UVP phosphoimager (Upland, CA).
Western blotting. HEK293 cells were seeded in 6 well tissue culture plates and
grown to 95% confluency in DMEM containing 10% FBS. The cells were serum deprived for
24 h prior to the treatment to reduce the basal levels of phospho-Met. Following serum
starvation, cocktails comprised of vehicle and HGF with/without 6-AH analogs were
prepared and pre-incubated for 30 min at room temperature. The cocktail was then added to
the cells for 10 min to stimulate the Met receptor and downstream proteins. Cells were
harvested using RIPA lysis buffer (Millipore; Billerica, MA) fortified with phosphatase
inhibitor cocktails 1 and 2 (Sigma-Aldrich; St. Louis, MO). The lysate was clarified by
centrifugation at 15,000 nx g for 15 min, protein concentrations were determined using the
BCA total protein assay (Pierce), and then appropriate volumes of the lysates were diluted
with 2x reducing Laemmli buffer and heated for ten min at 95° C. Samples containing
identical amounts of protein were resolved using SDS-PAGE (Criterion, BioRad
Laboratories), transferred to nitrocellulose, and blocked in Tris-buffered saline (TBS)
containing 5% milk for 1 h at room temperature. The phospho-Met antibody were added to
the blocking buffer at a final concentration of 1:1000 and incubated at 4° C overnight with
gentle agitation. The membranes were then washed several times with water and TBS (PBS,
0.05% Tween-20), a 1:5000 dilution of horseradish-peroxidase conjugated goat anti-rabbit
antiserum was added, and the membranes further incubated for 1 h at room temperature.
Proteins were visualized using the Supersignal West Pico Chemiluminescent Substrate
system (Pierce, Fenton, MO) and molecular weights determined by comparison to protein
ladders (BenchMark, Invitrogen, and Kaleidoscope, BioRad). Film images were digitized
and analyzed using a UVP phosphoimager.
Cell proliferation. 5000 MDCK cells were seeded into the wells of a 96 well plates
in 10% FBS DMEM. To induce cellular quiescence, the cells were serum deprived for 24 h
prior to initiating the treatments. Following serum starvation, 10 ng/ml HGF alone and with
various concentrations of 6-AH analogs or PBS vehicle were added to the media. The cells
were allowed to grow under these conditions for 4 days with a daily addition of 6-AH
analogs. On the fourth day, 1 mg/ml of 1-(4, 5-Dimethylthiazolyl) 3, 5-diphenylformazan
reagent (MTT, Sigma-Aldrich) prepared in PBS was added to the cells and incubated for 4 h.
Dimethyl sulfoxide diluted in a .01M glycine buffer was added to solubilize the cell
membranes and the absorbance of reduced MTT in the buffer was quantitated at 590 nm
using a plate reader (Biotek Synergy 2, Winooski, VT). HGF-dependent proliferation was
determined by subtracting the basal proliferation (in the absence of HGF) from total
proliferation rates in groups containing HGF.
Scattering assay. MDCK cells were grown to 100% confluency on the coverslips in
six-well plates and washed twice with PBS. The confluent coverslips were then aseptically
transferred to new six well plates containing 900 µl serum free DMEM. Norleual, Hinge
peptide, and/or HGF (20 ng/ml) were added to appropriate wells. Control wells received PBS
vehicle. Plates were incubated at 37°C with 5% CO for 48 h. Media was removed and cells
were fixed with methanol. Cells were stained with Diff-Quik Wright-Giemsa (Dade-Behring,
Newark, DE) and digital images were taken. Coverslips were removed with forceps and
more digital images were captured. Pixel quantification of images was achieved using Image
J and statistics were performed using Prism 5 and InStat v.3.05 (GraphPad; San Diego, CA).
Lung colony formation. Six to eight month old C57BL/6 mice were injected with
400,000 B16-F10 cells in 200 µl PBS by tail vein injection and subsequently received daily
intraperitoneal injections of either D-Nle-X-Cys-NH-(CH ) -CONH (10 µg/kg and
2 5 2
100µg/kg) or a PBS vehicle control. Two weeks later, mice were anesthetized and lungs
were perfused with PBS and removed. Photos were taken and lungs were solubilized in 1%
Triton x-100, 20 mM Tris, 0.15 M NaCl, 2 mM EDTA, and 0.02% sodium azide. Samples
were disrupted by sonication (Mixonix, Farmingdale, NY) and spun. The supernatant was
transferred to a 96 well plate and melanin absorbance at 410nm was measured using a plate
reader.
Statistics. Independent one-way analysis of variance (ANOVA) (InStat v.3.05 and
Prism 5) was used to determine differences among groups. Tukey-Kramar or Bonferroni’s
multiple comparison post-hoc tests were performed where necessary. Statistical comparisons
of two groups were determined using the two-tailed Student’s t-test (InStat v.3.05 and Prism
RESULTS
The AngIV analog D-Nle-Tyr-Ile-NH-(CH ) -CONH is more metabolically stable
2 5 2
than Norleual (Nle-Tyr-Leu-ψ-(CH -NH ) -His-Pro-Phe (SEQ ID NO: 1):The AngIV-
related peptidomimetic Norleual was previously shown to possess, anti-HGF/Met, anti-
angiogenic, and anti-cancer activities (Yamamoto et al., 2010). The presence of unprotected
peptide bonds at both the N- and C-terminal linkages predicts that Norleual should have poor
metabolic stability and rapid clearance for the circulation, properties that may limit its clinical
utility. In an attempt to overcome this limitation, a family of compounds, the 6-AH family
was designed and synthesized to offer defense against exopeptidases. Figure 22
demonstrates that as expected Norleual is unstable in heparinized blood while D-Nle-Tyr-Ile-
NH-(CH ) -CONH exhibited improved stability.
2 5 2
The AngIV analog D-Nle-Tyr-Ile-NH-(CH ) -CONH has a much longer
2 5 2
circulating half-life than Norleual (Nle-Tyr-Leu-ψ-(CH -NH ) -His-Pro-Phe (SEQ ID
NO: 1)):
As anticipated from the in-vitro blood stability data, D-Nle-Tyr-Ile-NH-(CH ) -CONH
2 5 2
exhibited an extended in vivo elimination half-life of 1012 min after IV injection in rats.
Other relevant pharmacokinetic parameters of D-Nle-Tyr-Ile-NH-(CH ) -CONH after a
2 5 2
single IV bolus dose are summarized in Table 5. Serum data were modeled using
WinNonlin® software to perform non-compartmental analysis. D-Nle-Tyr-Ile-NH-(CH ) -
CONH appeared to be extensively distributed outside the central blood compartment and/or
bound within the tissues as evidenced by its large volume of distribution (Vd). D-Nle-Tyr-Ile-
NH-(CH ) -CONH is not expected to be highly bound to plasma proteins according to
2 5 2
quantitative structure-activity relationship (QSAR) modeling (discussed below) and since
total recovery from serum was greater than 35 %. These results, which suggest that D-Nle-
Tyr-Ile-NH-(CH ) -CONH is likely to be relatively hydrophobic, are in agreement with the
2 5 2
outcome of QSAR modeling estimates generated by ADMET Predictor® that calculated an
octanol:water partition coefficient of 28.18 for D-Nle-Tyr-Ile-NH-(CH ) -CONH (Table 6).
2 5 2
Not surprisingly because of its stability, hydrophobic character, and small size, D-Nle-
Tyr-Ile-NH-(CH ) -CONH was predicted to be orally bioavailable. The P value represents
2 5 2 eff
the predicted effective human jejunal permeability of the molecule. The predicted P value
for D-Nle-Tyr-Ile-NH-(CH ) -CONH (1.53) is intermediate between the predicted P values
2 5 2 eff
for enalapril (1.25) and piroxicam (2.14), two orally bioavailable drugs. D-Nle-Tyr-Ile-NH-
(CH ) -CONH was also predicted to be 42.68 percent unbound to plasma proteins in
2 5 2
circulation, thus making it available for distribution into the tissues.
Also contributing to its slow removal from the blood was a lack of Phase I
metabolism for D-Nle-Tyr-Ile-NH-(CH ) -CONH . D-Nle-Tyr-Ile-NH-(CH ) -CONH
2 5 2 2 5 2
exhibited no detectable metabolism over 90 min in an in-vitro metabolism assay using rat
liver microsomes (data not shown). Together these data indicate that D-Nle-Tyr-Ile-NH-
(CH ) -CONH is more metabolically stable than Norleual, possesses an elongated half-life in
2 5 2
the circulation and penetrates tissue effectively. Overall these favorable pharmacokinetic
properties justify the mechanistic and therapeutic evaluation of D-Nle-Tyr-Ile-NH-(CH ) -
CONH and related molecules.
D-Nle-X-Ile-NH-(CH ) -CONH analogs bind HGF and compete with the H-
2 5 2
Hinge peptide for HGF binding:
Several members of the D-Nle-X-Ile-NH-(CH ) -CONH 6-AH family, were analyzed for the
2 5 2,
capacity to compete for H-Hinge binding to HGF. As will be evident below, members of the
6-AH family display a varied ability to block the biological action of HGF. As such, the HGF
binding properties of a selection of analogs with varying biological activity was assessed to
determine if there was a relationship between inhibitory activity and affinity for HGF. The
hypothesis that was put forth was that analogs are binding directly to HGF and affecting the
sequestration of HGF in an inactive form. To begin the evaluation of this idea, we used a H-
Hinge peptide as a probe to assess direct HGF binding of the peptides. The use of H-Hinge
to probe the interaction was based on the ability of H-Hinge to bind specifically and with
high affinity to HGF (Kawas et al., 2011). A competition study was initiated with several
derivatives of the D-Nle-X-Ile-NH-(CH ) -CONH family. This study demonstrated that
2 5 2
different analogs have variable abilities to bind HGF, and that the analogs showing
antagonism to HGF are acting as a Hinge mimics. D-Nle-X-Ile-NH-(CH ) -CONH
2 5 2
derivatives were found to compete with Hinge for HGF binding and exhibited a range of
-7 -10
affinities for HGF, with K s ranging from 1.37x10 - 1.33x10 M (Figure 23). As expected
it appears to be relationship between a compound’s ability to bind HGF and its capacity to
block dimerization and inhibit HGF-dependent activities (see Figures 25, 26, 27).
D-Nle-X-Ile-NH-(CH ) -CONH analogs block HGF Dimerization: Several reports
2 5 2
have shown that HGF needs to form homodimers and/or multimers, prior to its activation of
Met (Chirgadze et al., 1999; Gherardi et al., 2006). This dimer is arranged in a head to tail
orientation; the dimer interface comprises a central region, the hinge region that is important
for the proper dimer formation and orientation. A homologous sequence-conservation screen
against all possible transcripts that were independent of and not derived from angiotensinogen
looking for similarities to AngIV identified partial homology with the hinge region
(Yamamoto et al., 2010) of the plasminogen family of proteins, which include plasminogen
itself, its anti-angiogenic degradation product, angiostatin, and the protein hormones
heptocyte growth factor (HGF) and macrophage stimulating protein (MSP). Moreover, the
AngIV analog Norleual, which is a potent inhibitor of the HGF/Met system, was shown to
bind to HGF and block its dimerization (Kawas et al., 2011). This knowledge coupled with
the demonstration that some members of the 6-AH family bound with high affinity to the
hinge region of HGF led to the expectation that other active AngIV analogs, like 6-AH family
members, could be expected to inhibit HGF dimerization and that the ability of an individual
analog to bind HGF and inhibit HGF-dependent processes should be reflected in its capacity
to attenuate dimerization. The data in Figure 24 confirm this expectation by demonstrating
that D-Nle-Cys-Ile-NH-(CH ) -CONH and D-Nle-Tyr-Ile-NH-(CH ) -CONH , which bind
2 5 2 2 5 2
HGF with high affinity (Figure 23) and effectively attenuate HGF-dependent processes
(Figures 25, 26, 27 ) completely block HGF dimer formation. Conversely D-Nle-Met-Ile-
NH-(CH ) -CONH , which has low affinity for HGF (Figure 23) and exhibits little anti-
2 5 2
HGF/Met activity, is unable to block dimerization at the concentration tested. The D-Nle-Trp-
Ile-NH-(CH ) -CONH analog, which exhibits intermediate inhibition of dimerization,
2 5 2
predictably has a moderate affinity for HGF and a moderate ability to inhibit HGF-dependent
processes (Figures 25, 26, 27). Together these data confirm the expectation that active 6-AH
analogs can block dimerization and further that dimerization inhibitory potential of an analog
translates, at least qualitatively, to its capacity to block HGF-dependent processes.
D-Nle-X-Ile-NH-(CH ) -CONH analogs attenuates HGF-dependent Met
2 5 2
signaling:
After establishing that the 6-AH family members exhibit a range of HGF binding and
dimerization inhibitory profiles, we next determined whether these properties would parallel a
compound’s ability to inhibit Met signaling. Characteristic of tyrosine kinase-linked growth
factor receptors like Met is a requisite tyrosine residue auto-phosphorylation step, which is
essential for the eventual recruitment of various SH2 domain signaling proteins. Thus we
evaluated the ability of several 6-AH analogs to induce Met tyrosine phosphorylation. As
anticipated, the data in Figure 25 demonstrate that both D-Nle-Cys-Ile-NH-(CH ) -CONH
2 5 2
and D-Nle-Tyr-Ile-NH-(CH ) -CONH , which bind HGF with high affinity (Figure 23) and
2 5 2
effectively block its dimerization
(Figure 24) were able to block Met auto-phosphorylation. The D-Nle-Trp-Ile-NH-(CH ) -
CONH analog had intermediate inhibitory activity, and the D-Nle-Met-Ile-NH-(CH ) -
2 2 5
CONH analog showed no ability to effect on Met activation. Together, these data indicate
that the capacity of 6-AH analogs to inhibit HGF-dependent Met activation paralleled their
HGF binding affinity and their capacity to block dimerization.
D-Nle-X-Ile-NH-(CH ) -CONH analogs affect HGF/Met stimulated MDCK cell
2 5 2
proliferation:
Met activation initiates multiple cellular responses including increased proliferation and
motility, enhanced survival, and differentiation (Zhang and Vande Woude, 2003). As an
initial test of the ability of 6-AH family members to alter HGF-dependent cellular activity we
evaluated the capacity of several members of the family to modify the proliferative activity of
Madin-Darby canine kidney (MDCK) cells, a standard cellular model for investigating the
HGF/Met system (Stella and Comoglio, 1999). As seen in Figure 26 there is a wide range of
inhibitory activity against HGF dependent cellular proliferation. Similar to the results from
the binding and dimerization experiments the Cys and Tyr analogs exhibited marked
inhibitory activity. The Asp analog, which had not been evaluated in the earlier studies, also
2 2 2
exhibited pronounced inhibitory activity. The Trp , Phe , and Ser analogs all showed
inhibitory activity, albeit less than that observed with the most potent analogs. The decrease
in HGF-dependent MDCK proliferation below control levels for some compounds is not
surprising since the experiment was carried in 2% serum, which likely contains some level of
HGF. The Hinge peptide (KDYIRN), which represents the dimerization domain of HGF, was
included as a positive control. A recent study has demonstrated that Hinge binds to HGF with
high affinity blocking its dimerization and acting as a potent inhibitor of HGF-dependent
cellular activities including MDCK proliferation (Kawas et al., 2011).
D-Nle-X-Ile-NH-(CH ) -CONH analogs modify HGF/Met mediated cell scattering
2 5 2
in MDCK cells:
Cell scattering is the hallmark effect of HGF/Met signaling; a process characterized by
decreased cell adhesion, increased motility, and increased proliferation. The treatment of
MDCK cells with HGF initiates a scattering response that occurs in two stages. First, the cells
lose their cell-to-cell adhesion and become polarized. Second, they separate completely and
migrate away from each other. It is expected that if the 6-AH family members are capable of
inhibiting the HGF/Met system then they should be able to modify HGF dependent MDCK
cell scattering.
Figures 27 A & B indicate that those analogs that were previously found to block
HGF dimerization were effective inhibitor of HGF/Met mediated cell scattering in MDCK
cells, while those analogs with poor affinity for HGF were ineffective. Figure 28 shows a
correlation between the blockade of HGF dimerization and HGF binding affinity and the
ability to prevent MDCK cell scattering.
D-Nle-Cys-Ile-NH-(CH ) -CONH inhibits B16-F10 murine melanoma cell
2 5 2
migration and lung colony formation:
To evaluate the prospective utility of the 6-AH family members’ as potential therapeutics, we
examined the capacity of [D-Nle-Cys-Ile-NH-(CH ) -CONH ], an analog that exhibits a
2 5 2
strong inhibitory profile against HGF-dependent Met activation, to suppress the migratory
and lung colony-forming capacity of B16-F10 murine melanoma cells. B16 melanoma cells
over-express Met (Ferraro et al., 2006), and were chosen for these studies because Met
signaling is critical for their migration, invasion, and metastasis. As a final test for the
physiological significance of the 6-AH family blockade of Met-dependent cellular outcomes,
we evaluated the ability of D-Nle-Cys-Ile-NH-(CH ) -CONH to inhibit the formation of
2 5 2
pulmonary colonies by B16-F10 cells after tail vein injection in mice. Figure 29a illustrates
the inhibitory response that was observed with daily intraperitoneal injections at two doses
(10µg/kg/day and 100µg/kg/day) of [D-Nle-Cys-Ile-NH-(CH ) -CONH ]. Figure 29b
2 5 2
provides a quantitative assessment of pulmonary colonization by measuring melanin content,
which reflects the level of melanoma colonization. Together these data demonstrate that
treatment of melanoma cells with D-Nle-Cys-Ile-NH-(CH ) -CONH radically prevented lung
2 5 2
colonization and highlight the utility of the 6-AH analogs as anti-cancer agents.
DISCUSSION:
Recently interest has grown in developing therapeutics targeting the HGF/Met system.
At present this interest has been primarily driven by the realization that over-activation of the
HGF/c-Met system is a common characteristic of many human cancers (Comoglio et al.,
2008; Eder et al., 2009). The potential utility of anti-HGF/Met drugs, however, goes well
beyond their use as anti-cancer agents. For example, the recognized involvement of the
HGF/c-Met system in the regulation of angiogenesis (see review- supports the potential utility
of HGF/Met antagonists for the treatment of disorders in which control of tissue
vascularization would be clinically beneficial. These could include hyper-vascular diseases
of the eye like diabetic retinopathy and the wet type of macular degeneration. In both cases
anti-angiogenic therapies are currently in use (see review- Jeganathan, 2011). Anti-
angiogenics are also being examined as treatment options in a variety of other disorders
ranging from obesity where adipose tissue vascularization is targeted (Daquinag et al., 2011),
to chronic liver disease (Coulon et al., 2011), to psoriasis where topical application of anti-
angiogenic drugs is being considered (Canavese et al., 2010).
Currently the pharmaceutical industry is employing two general approaches to block
Met-dependent cellular activities (Eder et al., 2009; Liu X et al 2010). The first involves the
development of single-arm humanized antibodies to HGF (Burgess et al., 2006; Stabile et al.,
2008) or Met (Martens et al., 2006). The second approach utilizes “kinase inhibitors”, which
block the intracellular consequences of Met activation. These ‘kinase inhibitors” are small
hydrophobic molecules that work intracellularly to compete for the binding of ATP to the
kinase domain of Met thus inhibiting receptor autophosphorylation., 2002; Christensen et al.,
2003; Sattler et al., 2003). Despite the promise of the biologic and kinase-inhibitor
approaches, which are currently represented in clinical trials, both have limitations arising
from toxicity or specificity considerations and/or cost (Hansel et al., 2010; Maya, 2010).
A third approach, which our laboratory has been pursuing exploits a step in the
activation process of the HGF-Met system; namely the need for HGF to pre-dimerize before it
is able to activate Met. Thus we have targeted the dimerization process by developing
molecules that mimic the dimerization domain, the hinge region, with idea that they can act
as dominant negative replacements. Recent studies have validated this general approach
demonstrating that molecules designed around angiotensin IV (Yamamoto et al, 2010) or the
hinge sequence itself (Kawas et al., 2011) can bind HGF, block its dimerization, and
attenuate HGF-dependent cellular actions. The studies described herein represent a first step
toward producing useful therapeutics targeted at HGF dimerization. The primary focus of this
study was to improve the pharmacokinetic characteristics of a parent compound, Norleual
(Yamamoto et al., 2010) while maintaining biological activity. To this end we successfully
synthesized and evaluated a family of new molecules, the 6-AH family [D-Nle-X-Ile-NH-
(CH ) -COOH]. A subset of these molecules not only had improved metabolic stability and
circulating t but exhibited excellent in vitro and in vivo activity.
In addition to characterizing a new family of HGF/Met antagonists, this Example
demonstrates a qualitative relationship between the ability of a compound to bind HGF and
block HGF dimerization and its observed in vitro biological activity. Moreover these studies
provide initial structure-activity data and pave the way for more extensive evaluation. The
chemical modifications that were made at the N- and C-terminals of the AngIV molecule and
the resultant improvement in metabolic stability highlight the critical role played by
exopeptidases in the metabolism of AngIV-derived molecules. The demonstrated importance
of protecting the terminals to pharmacokinetic characteristics suggests numerous additional
synthetic approaches that may be applicable including the insertion of non-peptide linkages
(see Sardinia et al., 1994) between the first and second amino acids, the replacement of the N-
terminal amino acid with a non-α amino acid, and N-terminal acylation.
In sum these studies further validate the notion that targeting the dimerization domain
of HGF is an effective means of inhibiting the HGF/Met system. Further they demonstrate
that molecules with favorable pharmacokinetic characteristics can be produced thus
highlighting their clinical utility.
Table 5. WinNonlin® estimated pharmacokinetic parameters for D-Nle-Tyr-Ile-
NH-(CH ) -CONH after intravenous administration in adult male Sprague-Dawley
2 5 2
rats Mean+/- SEM; n = 5. AUC = area under the curve. Vd= volume of distribution. Cp =
initial concentration of drug in serum. t = biological half-life. KE= rate of elimination. CL=
clearance rate.
Pharmacokinetic Parameter D-Nle-Tyr-Ile-NH-(CH ) -CONH
2 5 2
(Mean ± SEM)
AUC0-∞ (min.ng/mL) 692.5 ± 293.2
Vd (L/kg) 104186.8 ± 65034.3
Cp (ng/mL) 68.2 ± 32.2
t1/2 (min) 1012.0 ± 391.4
KE (min-1) 0.001 ± 0.0002
CL (L/min/kg) 58.3 ± 15.6
Table 6. Predicted physiochemical properties of D-Nle-Tyr-Ile-NH-(CH ) -CONH . The
2 5 2
physiochemical properties of D-Nle-Tyr-Ile-NH-(CH ) -CONH were estimated following
2 5 2
modeling with ADMET Predictor® software. LogP is the octanol:water partitioning
coefficient. P is the predicted effective human jejunal permeability. P is the approximate
eff avg
average intestinal permeability along the entire human intestinal tract. Pr is the percent
Unbnd
unbound to plasma proteins.
Physicochemical Property Predicted Value
logP 1.45
P 1.53
P 0.39
Pr 42.68
Unbnd
While the invention has been described in terms of its preferred embodiments, those
skilled in the art will recognize that the invention can be practiced with modification within
the spirit and scope of the appended claims. Accordingly, the present invention should not be
limited to the embodiments as described above, but should further include all modifications
and equivalents thereof within the spirit and scope of the description provided herein.
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless specifically stated otherwise,
reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of
the common general knowledge in the art.
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Claims (46)
1. A hepatocyte growth factor (HGF) mimic having the general formula: R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, 10 lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and 15 n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
2. The HGF mimic of claim 1 wherein R is tyrosine.
3. N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide.
4. A composition, comprising: at least one hepatocyte growth factor (HGF) mimic having the general formula: R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, 5 phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; 10 R is isoleucine; and n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds; and a carrier, said HGF mimic being dissolved or distributed in said carrier.
5. A composition comprising N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide and a carrier, said N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide being dissolved or distributed in said carrier. 20
6. Use, in the manufacture of a medicament for enhancing cognitive function or treating or preventing cognitive dysfunction in a subject, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, 5 lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and 10 n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
7. The use of claim 6 wherein said medicament is to be administered multiple times over a 15 period of time.
8. The use of claim 7 further comprising the steps of testing cognition of said subject during said period of time, and adjusting an amount of said HGF mimic to be administered based on test results.
9. Use, in the manufacture of a medicament for expanding synaptic connectivity and/or bringing about neuronal replacement in a subject, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; 5 R is isoleucine; and n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
10 10. The use of claim 9 wherein said subject has suffered spinal cord trauma.
11. Use, in the manufacture of a medicament for treating dementia, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; 20 R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and n ranges from 3-6; 25 and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
12. Use, in the manufacture of a medicament for providing neuroprotection or inducing neuroregeneration, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 5 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, 10 aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide 15 bonds or reduced peptide bonds.
13. Use, in the manufacture of a medicament for improving cognitive function in individuals with normal cognitive capacities thereof, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, 5 lysine and valine; R is isoleucine; and n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
14. Use, in the manufacture of a medicament for treating cancer, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; 20 R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and n ranges from 3-6; 25 and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
15. Use, in the manufacture of a medicament for treating diabetes, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where 5 R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, 10 lysine and valine; R is isoleucine; and n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
16. Use, in the manufacture of a medicament for treating fibrotic disease, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 3 2 n 2 1 2 3 20 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and 5 n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
17. The use of claim 16 wherein said fibrotic disease is selected from cardiac, pulmonary, 10 renal, or hepatic fibrosis.
18. Use, in the manufacture of a medicament for treating vascular insufficiency, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; 20 R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and n ranges from 3-6; 25 and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
19. The use of claim 18 wherein said vascular insufficiency is selected from deep vein thrombosis or coronary artery occlusion.
20. Use, in the manufacture of a medicament for facilitating wound healing, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 2 n 2 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, 10 lysine, norvaline, ornithine, and s-benzyl cysteine; R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and 15 n ranges from 3-6; and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
21. Use, in the manufacture of a medicament for retarding or reversing the 20 hypervascularization of the eye, of one or more hepatocyte growth factor mimics having the general structural formula R -R -R -NH-(CH) -C-NH 1 2 3 2 n 2 1 2 3 where R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine, norvaline, ornithine, and s-benzyl cysteine; 5 R is an amino acid selected from the group consisting of tyrosine, phenylalanine, aspartic acid, arginine, isoleucine, serine, histidine, glycine, cysteine, methionine, tryptophan, lysine and valine; R is isoleucine; and n ranges from 3-6; 10 and wherein covalent bonds 1, 2 and 3 are selected from the group consisting of peptide bonds or reduced peptide bonds.
22. The use of claim 21 wherein said hypervascularization of the eye is in a disease selected from diabetic retinopathy or macular degeneration.
23. The use of any one of claims 6 to 12 and 14 to 22 wherein said medicament comprises a therapeutic amount of said one or more hepatocyte growth factor mimics.
24. The use of claim 13 wherein said medicament comprises a facilitory amount of said one 20 or more hepatocyte growth factor mimics.
25. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for enhancing cognitive function or treating or preventing cognitive dysfunction in a subject.
26. The use of claim 25 wherein said medicament is to be administered multiple times over a period of time.
27. The use of claim 26 further comprising the steps of testing cognition of said subject 30 during said period of time, and adjusting an amount of said N-hexanoic-L-tyrosine-L- isoleucine-(6)-aminohexanoic amide to be administered based on test results.
28. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for expanding synaptic connectivity and/or bringing about neuronal replacement in a subject. 5
29. The use of claim 28 wherein said subject has suffered spinal cord trauma.
30. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for treating dementia. 10
31. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for providing neuroprotection or inducing neuroregeneration.
32. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for improving cognitive function in individuals with normal cognitive 15 capacities thereof.
33. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for treating cancer. 20
34. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for treating diabetes.
35. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for treating fibrotic disease.
36. The use of claim 35 wherein said fibrotic disease is selected from cardiac, pulmonary, renal, or hepatic fibrosis.
37. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture 30 of a medicament for treating vascular insufficiency.
38. The use of claim 37 wherein said vascular insufficiency is selected from deep vein thrombosis or coronary artery occlusion.
39. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for facilitating wound healing.
40. Use of N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide in the manufacture of a medicament for retarding or reversing the hypervascularization of the eye.
41. The use of claim 40 wherein said hypervascularization of the eye is in a disease selected 10 from diabetic retinopathy or macular degeneration.
42. The use of any one of claims 25 to 31 and 33 to 41 wherein said medicament comprises a therapeutic amount of said N-hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide. 15
43. The use of claim 32 wherein said medicament comprises a facilitory amount of said N- hexanoic-L-tyrosine-L-isoleucine-(6)-aminohexanoic amide.
44. A hepatocyte growth factor mimic of any one of claims 1 to 3 substantially as herein described with reference to any example thereof and with or without reference to the 20 accompanying figures.
45. A composition of claim 4 or 5 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures. 25
46. The use of any one of claims 6 to 43 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161471124P | 2011-04-02 | 2011-04-02 | |
US201161471122P | 2011-04-02 | 2011-04-02 | |
US61/471,124 | 2011-04-02 | ||
US61/471,122 | 2011-04-02 | ||
PCT/US2012/031815 WO2012138599A2 (en) | 2011-04-02 | 2012-04-02 | Hepatocyte growth factor mimics as therapeutic agents |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ616210A NZ616210A (en) | 2015-07-31 |
NZ616210B2 true NZ616210B2 (en) | 2015-11-03 |
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