WO2013112765A1

WO2013112765A1 - Effect of orally administered dipterinyl calcium pentahydrate (dcp) on oral glucose tolerance in dio mice

Info

Publication number: WO2013112765A1
Application number: PCT/US2013/023027
Authority: WO
Inventors: Phillip Moheno; Dietmar Fuchs; Svetlana E. NIKOULINA
Original assignee: Sanrx Pharmaceuticals, Inc.
Priority date: 2012-01-24
Filing date: 2013-01-24
Publication date: 2013-08-01

Abstract

Provided herein are methods of treating diabetes comprising administering to a subject a therapeutically effective amount of dipterinyl calcium pentahydrate (DCP). Dipterinyl calcium pentahydrate (DCP) is a molecular entity whose structure is based upon the endogenous substance pterin, has effects on glucose tolerance and other measures in vivo were determined in mice fed a high-fat diet to induce obesity and insulin resistance. DCP was found to significantly improve oral glucose tolerance (OGTT) in female DIO mice,C57BL/67.

Description

EFFECT OF ORALLY ADMINISTERED DIPTERINYL CALCIUM PENTAHYDRATE (DCP) ON ORAL GLUCOSE TOLERANCE IN DIO MICE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application Serial No. 61/590,322, filed January 24, 2012, which is hereby incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

[0002] This application provides dipterinyl calcium pentahydrate ( DCP ) a novel therapeutic for Type 2 diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0004] Figure la: Female DIO Mice, C57BL/6J, fasting blood glucose levels during DCP dosing

[0005] Figure lb: Female DIO Mice, C57BL/6J, body weights during DCP dosing

[0006] 24 female DIO mice, C57BL/6J were used. The mice were fed ad libitum a high-fat diet starting at six weeks of age until 18 weeks. For 21 days afterwards, the test article, dipterinyl calcium pentahydrate (DCP) in 0.4% carboxymethylcellulose, was administered daily by oral gavage at 0, 7, 21, or 63 mg/(kg d) to four groups of six mice each. During the DCP gavaging period three mice were lost due to gavaging trauma: one control and two from the 63 mg/(kg d) group.

[0007] DETAILED DESCRIPTION OF THE INVENTION

Type 2 diabetes, often called non-insulin dependent diabetes, is the most common form of diabetes, affecting 90% - 95% of the 21 million people. Obesity is a disease characterized by chronic inflammation and the progressive loss in systemic insulin sensitivity. Obesity and type 2 diabetes Chronic inflammation is currently considered as a common feature. One of the mechanisms by which chronic inflammation might trigger and/or maintain the development of is transcriptional induction of indoleamine 2,3-dioxygenase (IDO), rate-limiting enzyme of tryptophan (TRY)-kynurenine (KYN) pathway. It has been determined that elevated tryptophan levels can lead to increased formation of formate and indolyl metabolites, several of which inhibit the degradation of histamine, potentially leading to eosinophilia-myalgia syndrome (EMS) and diabetes.

High inflammatory cytokines levels and other inflammatory markers, also, represent a basic cause and consequence of obesity, type 2 diabetes, and comorbidities. Modulation of the levels of anti-inflammatory and pro-inflammatory cytokines is a strategy of therapeutic intervention in the treatment of type 2 diabetes and metabolic disease.

Dipterinyl calcium pentahydrate (DCP) is a molecular entity whose structure is based upon the endogenous substance pterin, has effects on glucose tolerance and other measures in vivo were determined in mice fed a high- fat diet to induce obesity and insulin resistance. DCP was found to significantly improve oral glucose tolerance (OGTT) in female DIO mice,C57BL/67.

DCP works through a broad immunomodulatory mechanism involving a key immuno-inhibitory enzyme, indoleamine 2,3-dioxygenase (IDO) which DCP appears to modulate to a

"homeostatic" level. DCP inhibits IDO in some systems, and DCP promotes IDO in other systems, DCP also increases the plasma cytokines IL-12 and IL-4, and chemokine GM-CSF, while decreasing IL-6 and MCP-1. DCP potentiated monocyte antimycobacterial activity by induction of the C-C chemokine ΜΙΡ-Ιβ, and inducible nitric oxide synthase 2.

In the present study the female DIO mice , C57BL/6J was chosen because they were presumed to have higher levels of indoleamine 2,3-dioxygenase (IDO) than males based upon an earlier study.

In an effort to evaluate oral glucose tolerance (OGTT), DCP was administrated daily by oral gavage at 0.7 mg/kg, 21 mg/kg, 63mg/kg and control. Each group of the study was formed of six mice.

[0008] Materials and methods

[0009] Test substances

[0010] Dipterinyl calcium pentahydrate (DCP) synthesis:

[0011] Pure pterin (81.7 mg, 0.5 mmol) was dissolved in H20 (50 ml) and 0.1 N NaOH

(6 ml) and CaC12 * 2H20 (36.7 mg, 0.25 mmol) was added to the clear solution with stirring (pH 10.93). A yellowish precipitate was formed within a few minutes. Stirring was continued for 1 day and then the precipitate collected and dried in a vacuum desiccator to give 75 mg. The elemental analysis is consistent with

(C6H4N50)2Ca^> 5H20 (MW 454.4).

[0012] Calc. C 31.74 H 4.00 N 30.85

[0013] Found C 31.22 H 3.97 N 29.83 [0014] The comparison of the extinctions of the UV spectra of pterin and

(C6H4N50)2Ca^> 5H20 taken at pH 13 show the following:

[0015] Pterin: 223 nm (8,700), 250 nm (21,380), 357 nm (8,510)

[0016] (C6H4N50)2Ca^> 5H20: 223 nm (14,450), 250 nm (39,810), 357 nm

(13,490)

[0017] DCP in suspension in 0..4 % carboxymethylcellulose

[0018] In Vivo Testing

24 female DIO mice, C57BL/6J ⁷ (000664, B6; Jackson Labs, Sacramento, CA) were used because they were presumed to have higher levels of indoleamine 2,3-dioxygenase (IDO) than males based upon an earlier study. ² The mice were fed ad libitum a high- fat diet (60 kcal, Research Diets Inc. D12492i) starting at six weeks of age until 18 weeks. For 21 days afterwards, the test article, dipterinyl calcium pentahydrate (DCP) in 0.4%

carboxymethylcellulose, was administered daily by oral gavage at 0, 7, 21, or 63 mg/(kg d) to four groups of six mice each. During the DCP gavaging period three mice were lost due to gavaging trauma: one control and two from the 63 mg/(kg d) group. Two of the collected plasma samples collected after DCP dosing were inadvertently not labeled and therefore excluded from the analyses: one from the 21 mg/(kg d) group and one from the 63 mg/(kg d).

Measurements

Blood glucose measurements were taken from fasted animals twice weekly as

Animals were fasted for 6 hours prior to blood collections.

Blood (approximately 10 to 20 μί) was collected via tail snip, and subsequent bleeds by removing the scab. Additional bleeds were done by snipping the tail and then removing the scab, until 2 mm of the tail tip was removed. Additional bleeds were then done via tail pricks (i.e., sticking the vein or artery with a needle) from all animals beginning just prior to group assignment. Blood glucose was measured using the One Touch Ulta 2 Blood Glucose Monitoring System (LifeScan, a Johnson & Johnson company).

During the 21 day DCP dosing period, blood was collected 2 hours post-dose on days 1, 3, 5, 9, 10, 13, 17, and 21. 4) On Day 21 after DCP administration, an oral glucose tolerance test (OGTT) was carried out measuring pre-challenge blood glucose, and 30, 60, 90, and 120 minutes post-challenge with 2 g/kg glucose.

Plasma samples were collected by cardiac puncture on Day 21 after DCP administration. The plasma IDO metabolites, tryptophan and kynurenine, were measured by high-performance liquid chromatography (HPLC) ⁸ and the following cytokines and

chemokines; GM-CSF, IFNy, IL-la, IL-Ι β, IL-4, IL-6, IL-10, IL-12(p40), IL-12(p70), IL-13, MCP-1 , RANTES, and TNFa were measured by EMD Millipore (St. Charles, MO) using the Mouse Cytokine / Chemokine Magnetic Bead Panel Kit (96- Well Pate Assay #MCYTOMAG- 70K) (Luminex Corporation). Reported precisions for the overnight protocol for these cytokines and chemokines are: Intra-assay %CV < 4.9, Inter-assay %CV < 12.4.

Statistics

[0019] Standard and repeated measures analyses of variance (ANOVAs), and stepwise regression of the data were carried out using the SPSS Graduate Pack 15.0 (2006) and IBM SPSS Statistics v.19 for Windows (2010). The Test of Homogeneity of Variances was carried out to determine the appropriate Contrast Tests to be used in the Oneway ANOVAs. Statistical probability less than .05 (p < .05) was used to establish significance. During the 21 -day DCP dosing period there was no significant difference in blood glucose levels among the four treatment groups of 0, 7, 21, or 63 mg/(kg d) DCP as determined by repeated measures AN OVA (Analysis of Variance).

Results

Figure la and Figure lb that during the 21 day DCP dosing, there was no significant diffrerence in blood glucose or body weights among the four treatement groups. No toxicity was observed, Figure 1 b shows no statistically significant body weight change with 21 day dosing with DCP.

ANVOAs for the OGTT/AUC, plasma IDO metabolite, and cytokine/chemokine measures are given in Table 1 showing a significant 50% OGTT/AUC decrease for the mice treated with 7 mg/(kg d) DCP (p < .05).

Four variables were identified by stepwise regression to yield the following OGTT/AUC linear regression (eq. 1) (R² = .571 ; p = .013; see Table 2):

OGTT/AUC = 0.009 DCP³ + 31.178 DCP² - 574.513 DCP + 29.828 Trp + 1935.382 where DCP = DCP dosage in mg/(kg d), and Trp = mM plasma tryptophan. From this equation it can be predicted that lowered plasma tryptophan levels are expected to improve (i.e., decrease) OGTT/AUC measures.

The DCP dose corresponding to the minimum (least diabetic) OGTT/AUC values can be determined by setting the first derivative of eq. 1 equal to 0:

0 = d(OGTT/AUC)÷d(DCP) = 0.027 DCP² + 62.356 DCP - 574.513 which can be solved for DCP by using the quadratic formula:

DCP = -62.356 ± [(62.356)² - 4(O.027Y-574.513^^½ = -62.356 ± 62.852 = 0.496/0.054

2(0.027) 0.054

= 9.19 mg/(kg d) DCP

This DCP dosage calculated from the regression equation corresponds very well with the experimentally derived value of 7 mg/(kg d) from Table 1 for the anti-diabetic dosage giving the minimum OGTT/AUC.

From the Table 1 values interestingly, the plasma IL-6 pattern from Table 1, though non-significant, resembles the significant (p < .05) OGTT/ AUC pattern in that the lowest values for IL-6 and OGTT/AUC both correspond to a DCP dose of 7 mg/(kg d).

Investigation into the role of IL-6 in diabetes Type 1 and Type 2, and its role in the immune system generally in Type 2 diabetes.

Of particular interest is the finding that plasma tryptophan enters into the significant (p = .013) multivariate regression predicting the OGTT/AUC response (Table 2). that elevated tryptophan levels can lead to increased formation of formate and indolyl metabolites, several of which inhibit the degradation of histamine, potentially leading to eosinophilia- myalgia syndrome (EMS) and diabetes. In this regard, 98% of surveyed EMS patients from the US reported having used L-tryptophan-containing products prior to the onset of illness at a median dosage of 1,500 mg/day. ¹¹ This human dose of 1,500 mg / 70 kg = 21 mg/kg

corresponds to a mouse dosage of 264 mg/kg based upon body surface area allometrics. 264 mg/kg x [mmol Trp / 204 mg Trp] x [kg / 0.7 i] = 1.85 mM Trp, the estimated corresponding mouse plasma/tissue level increase, is well within the Table 1 SEM values for DIO mouse plasma Trp.

Table 1

One-way Analyses of Variance (ANOVAs) of Oral Glucose Tolerance Test Area-Under- Curve (OGTT/AUC); plasma Indoleamine 2,3-dioxygenase (IDO) metabolites tryptophan (Trp), kynurenine (Kyn), and calculated Trp/Kyn ratio; and certain cytokines/chemokines

**** /> = .001

For all DCP dosages For contrast

tested tests versus

0 mg/(kg d)

DCP

Table 2: Multivariate Linear Regression of Oral Glucose Tolerance Test Area-Under-

Curve (OGTT/AUC)

Trp, Kyn, Kyn/Trp, GM-CSF, ΙΚΝγ, IL-l , IL-Ιβ, IL-4, IL-6, IL-10, IL-12(p40), IL- 12(p70), IL-13, MCP-1, RANTES, and TNFa were tested by Stepwise Regression as predictors of OGTT/AUC. The selected variables were DCP³, DCP², DCP, and Trp (mM), where DCP is dosage in mg/(kg d). R² = .571; /? = .013 for the regression:

OGTT/AUC = 0.009 DCP³ + 31.178 DCP² - 574.513 DCP + 29.828 Trp + 1935.382

Coefficients'

a. Dependent Variable: OGTT/AUC

[0020] Dipterinyl calcium pentahydrate (DCP) was tested as a novel therapeutic for Type 2 diabetes. Female DIO mice, C57B/6J, fed a high-fat diet were administered DCP in 0.4% carboxymethylcellulose for 21 days. Blood glucose was followed during the dosing period, and an oral glucose tolerance test (OGTT) was carried out on day 21, along with measurements of plasma indoleamine 2,3-dioxygenase (IDO) metabolites (tryptophan and kynurenine), nitrite, and certain cytokines and chemokines. 7 mg/(kg d) DCP reduced OGTT/AUC (area under OGTT curve) by 50% (p < .05). A significant multivariate regression (p = .013; R2 = .571) of OGTT/AUC was derived from DCP dosage and plasma tryptophan. Elevated tryptophan, likely from diet and/or decreased IDO activity, was found to correlate with higher OGTT/AUC diabetic measures, possibly via inhibition of histamine degradation. Thus, an optimum dose of 7 mg/(kg d) DCP significantly improved the OGTT diabetic state in these female DIO mice.

[0021] Provided herein are methods wherein oral administration of DCP can improve glucose tolerance by normalizing the levels of pro-inflammatory cytokines which are elevated in obesity and type 2 diabetes. Effects on glucose tolerance and other measures in vivo were determined in mice fed a high- fat diet to induce obesity and insulin resistance. DCP was found to significantly improve oral glucose tolerance (OGTT) as determined by 2-hour area under curve (AUC) comparisons.

[0022] A series of ANVOAs for the OGTT/AUC, plasma IDO metabolite, and cytokine/chemokine measures is given in Table 1 showing a significant 50% AUC decrease for the mice treated with 7 mg/(kg d) DCP (p < .05).

[0023] Four variables were identified by stepwise regression to yield the following

OGTT/AUC regression (eq. 1) (R2 = .571; p = .013; see Table 2):

[0024] OGTT/AUC = 0.009 DCP3 + 31.178 DCP2 - 574.513 DCP + 29.828 Trp + 1935.382

[0025] where DCP = DCP dosage in mg/(kg d), and Trp = mM plasma tryptophan. From this equation it can be predicted that lowered plasma tryptophan levels resulting from diet and/or increased IDO activity are expected to improve (i.e., decrease) OGTT/AUC measures.

[0026] From the values for Trp (tryptophan), Kyn (kynurenine), and the calculated

Kyn/Trp ratio, it appears likely that IDO activity is not involved with the anti-diabetic

OGTT/AUC minimum at 7 mg/(kg d) DCP. Interestingly, the DCP dose vs. plasma IL-6 pattern from Table 1, though non-significant, resembles the significant (p < .05) DCP dose vs. OGTT/ AUC levels where the lowest values for IL-6 and OGTT/AUC both correspond to a DCP dose of 7 mg/(kg d). Investigation into the role of IL-6 in diabetes Type 1 and Type 2 [5], and into the immune system generally in Type 2 diabetes [4] has been called for.

[0027] The nitrite profile, though significant overall, did not yield significant individual contrasts vs. control. One study [8] has found that dietary inorganic nitrate can reverse features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice.

[0028] In conclusion, we find that:

[0029] 1) DCP significantly (p < .05) improves oral glucose tolerance at 7 mg/(kg d) in female DIO mice.

[0030] 2) A significant (p = .013) multiple regression, in terms of DCP3, DCP2, and DCP dosages, and plasma tryptophan levels, can be constructed, which explains 57% of the

OGTT/AUC variance in this measure of glucose tolerance in the DIO mice. [0031] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method of treating diabetes comprising administering to a subject a therapeutically effective amount of dipterinyl calcium pentahydrate (DCP).

2. The method of claim 1 wherein DCP is administered orally.

3. The method of claims 1 wherein DCP is administred as single active agent.

4. The method of claim 1 wherein DCP is administered in combination with a second antidiabetic medication.

5. The method of claim 4 wherein the second anti-diabetic medication is one or more of insulin; sensitizer agents; secretagogues; alpha glucosidase inhibitors; peptide analogues; and natural substances.