WO2020105027A1

WO2020105027A1 - Method and composition for preventing or treating diabetic and non-diabetic ketoacidosis using glycerol

Info

Publication number: WO2020105027A1
Application number: PCT/IL2019/051207
Authority: WO
Inventors: Hay Dvir
Original assignee: The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center)
Priority date: 2018-11-22
Filing date: 2019-11-05
Publication date: 2020-05-28
Also published as: IL263191A

Abstract

The present invention provides a pharmaceutical composition and method for preventing or treating diabetic ketoacidosis by administering an effective amount of glycerol, alone or with other agents.

Description

METHOD AND COMPOSITION FOR PREVENTING OR TREATING

DIABETIC AND NON-DIABETIC KETOACIDOSIS USING GLYCEROL

Field of the Invention

The present invention relates to methods for preventing or treating ketoacidosis by administering a therapeutically effective amount of a pharmaceutical composition comprising glycerol. More specifically, the invention relates to intravenous administration of glycerol for treating diabetic ketoacidosis (DKA).

Background of the Invention

Diabetes mellitus (DM) is a metabolic disorder of global health concern that develops due to lack or insufficient insulin activity. Since insulin is in charge of signaling for the glucose cellular uptake from the blood, its reduced activity results in high-blood glucose levels with potentially low-intracellular levels. Reduced insulin signaling also enhances adipose lipolysis and fat mobilization in the circulation in the form of fatty acids (FA). Depending on the hepatic energy balance, the uptake of circulating FA by the liver can lead to complete FA oxidative breakdown, or to partial FA breakdown. Intermediate products of the partially degraded FA serve as precursors for ketone bodies (KB) synthesis, a process that is also stimulated at low insulin signaling. Although secretion of KB to the circulation provides an alternative energy supply, at high concentration they acidify the blood (ketoacidosis) and lead to severe neurological consequences.

Low intracellular glucose levels limit the oxidative capacity of the liver for complete FA oxidation, thereby diverting more FA breakdown products for hepatic ketogenesis. Therefore DM patients, who are prone to develop very low hepatic glucose levels, are at risk of developing diabetes ketoacidosis (DK) - a life-threatening condition that can develop rapidly within 24 hours.

Patients with type 1 diabetes are more prone to developing DKA as a result of missed or inadequate insulin therapy, illness, infection or physical or emotional trauma. During an infection or illness the body produces hormones, such as adrenaline and cortisol, which counter the effect of insulin, thereby triggering DKA. Occasionally, type 1 diabetes is first diagnosed following symptoms of DKA. Type 2 diabetes patients may also develop DKA, though this is less common and usually not as severe as in type 1 diabetic patients.

Another type of ketoacidosis is alcoholic ketoacidosis caused by prolonged alcoholism. In itself, alcohol can be utilized for hepatic KB synthesis. But in case of alcoholism, consumption leads to dehydration and blocks the first step of gluconeogenesis, so that the body cannot synthesize enough glucose. As a result, more FA are only partially degraded in the liver, consequently promoting ketogenesis and increase in KB in the blood.

Ketoacidosis often requires admission to an emergency room for fluid and electrolyte replacement, such as sodium, potassium and chloride, as well as insulin therapy, which is less efficient in patients with insulin resistance, such as type II patients.

It would therefore be extremely important, and this is an object of the present invention, to provide an insulin-independent method for treating ketoacidosis.

Recently, a new class of promising drugs for glucose and metabolic control in diabetes were developed based on inhibiting sodium-glucose cotransporters (called also sodium- glucose linked transporters or SGLT). However, despite their significant advantages in conferring weight loss and cardiometabolic benefits, SGLT inhibitors (SGLTi) have been associated with increased rates of DKA, often with near-normal glucose levels, which is of an alarming concern for type 1 diabetes as delaying diagnosis and treatment of this emergent condition. Therefore, the development of SGLTi-adjunctive therapy against DKA may offer type 1 diabetes patients the metabolic benefits of SGLTi therapy, in a safer and more effective manner.

It was found that glycerol intravenous treatment of hyperketonemia and hypoglycemia in sheep is capable of reversing both conditions. Therefore, glycerol can be used to reduce blood ketone levels, and therefore should help in treatment and prevention of DKA, either independently or as an SGLTi-adjunctive therapy.

Glycerol (also known as glycerine or glycerin) is a natural organic precursor for synthesis of both carbohydrates and fats. Glycerol has many applications in the food industry as well as medical applications. In pharmaceuticals, glycerol is used as an excipient (pharmacologically inactive carrier substance) and is also used for reducing intraocular pressure in people with glaucoma.

One object of the invention is to provide efficient solutions to patients suffering from ketoacidosis. Once administered, glycerol is efficiently taken up by the liver, with little or no dependency on insulin function (unlike glucose). In the liver, glycerol is rapidly utilized for gluconeogenesis, thus replenishing the intracellular glucose shortage - the main trigger of DKA. Glycerol IV administration was, therefore, very efficient in reducing hyperketonemia (high blood KB) in ketotic sheep.

Another advantage of administering glycerol lies in the ability to infuse glycerol into the blood at relatively high doses to replenish intracellular carbohydrate shortage at relatively low risk of spiking blood glucose or increasing blood lactate, both of which may aggravate DKA.

One aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of glycerol for use in treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis.

Another aspect of this invention relates to a pharmaceutical composition comprising a therapeutically effective amount of glycerol for use as a SGLTi-adjunctive therapy in preventing, ameliorating, reducing or delaying the onset of ketoacidosis, particularly in type 1 diabetes patients. A further aspect of the invention is the provision of a method for treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis by administering a therapeutically effective amount of pharmaceutical composition comprising glycerol. Further purposes and advantages of this invention will appear as the description proceeds.

Summary of the Invention

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of glycerol for use in treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis. According to some embodiments, the ketoacidosis is Diabetic ketoacidosis (DKA).

According to some embodiments, the amount of glycerol in the composition is between 5% and 22%. In yet some further embodiments, the composition is administered intravenously.

Another aspect of the invention provides a method for treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis by administering a therapeutically effective amount of pharmaceutical composition comprising glycerol. In some of these embodiments, the ketoacidosis is Diabetic ketoacidosis (DKA). In some of these embodiments, the amount of glycerol in the composition is between 5% and 22%. In yet some further embodiments, the composition is administered intravenously.

In a preferred embodiment of the invention, the pharmaceutical composition comprising a therapeutically effective amount of glycerol is administered simultaneously with SGLTi (sodium-glucose linked transporters inhibitors).

Brief Description of the Drawings

- Figure 1 - b-hydroxybutyrate (BHBA) response to the propylene glycol (PG) and the glycerol treatments. BHBA is the most common KB to measure in the blood. Analysis shows no significant effect of treatment, a significant effect of time (P< 0.0001), a treatment by time interaction (P< 0.0001) and an individual animal effect (P< 0.026). **P< 0.01. SEM = 0.03. Values are Mean ± SE. Both PG and glycerol are very effective in reducing plasma KB.

- Figure 2 - Glucose response to the PG and the glycerol treatments. Analysis shows an effect of: treatment (P< 0.0031), time (P< 0.0001), treatment by time interaction (P< 0.0001), individual animal effect (P< 0.037). *P< 0.05, **P< 0.01, ***P< 0.0001. SEM = 0.974. Values are Mean ± SE. Glycerol is significantly more gluconeogenic.

- Figure 3 - Lactate response to the PG and the glycerol treatments. Analysis shows an effect of: treatment P< 0.0001, time P< 0.0001, a treatment-time interaction P< 0.0001, individual animal P< 0.021. **P< 0.01, ***P< 0.0001. SEM = 13.2. Values are Mean ± SE. PG stimulated plasma lactate significantly while glycerol did not.

- Figure 4 - Insulin response to the PG and the glycerol treatments. Analysis shows an effect of: treatment P<0.0009, time P< 0.0001, treatment-time interaction P = 0.0312, individual animal P< 0.0001. **P< 0.01. SEM = 0.01. Values are Mean ± SE. Glycerol stimulated plasma insulin significantly better than PG, likely secondary to its conversion to glucose.

- Figure 5 - Non-esterified fatty acids (NEFA) response to the propylene glycol and the glycerol treatments. Analysis shows an effect of: treatment P<0.026, time P< 0.0001, treatment-time interaction P< 0.0001, individual animal P<0.0521. **P< 0.01, ***p< 0.0001. SEM = 53. Values are Mean ± SE. Both PG and glycerol reduce adipose lipolysis, but the response to glycerol was significantly faster.

- Figure 6. Responses of liver and tissue damage biomarkers to PG and glycerol IV treatments. Figure 6A - Lactate dehydrogenase (LDH) over time from injection (P< 0.0001). Analysis shows an effect of time P< 0.0001, treatment-time interaction P< 0.0001, effect of individual animal P< 0.037. *P< 0.05, ***P< 0.0001. SEM = 25.3. Figure 6B - aspartate aminotransferase (AST) over time from injection (P< 0.01), Analysis shows an effect of time (P< 0.0001) and treatment-time interaction (P< 0.0001). **P< 0.01. SEM = 10.7. Figure 6C - Alanine aminotransferase (ALT) over time from injection (P< 0.0001), treatment-time interaction (P < 0.0001) and effect of individual animal (P < 0.0103). *P< 0.05, **P< 0.01. SEM = 1.3. Values are Mean ± SE. Unlike glycerol, PG shows indication for liver or tissue damage. - Figure 7. Plasma Bilirubin as a function of time from the propylene glycol and glycerol treatments. Figure 7A - Total bilirubin. Analysis shows an effect of treatment P< 0.0018, time P< 0.0093, treatment-time interaction P< 0.0001, individual animal P< 0.0198. *P< 0.05, **P< 0.01, ***P< 0.0001. SEM = 0.192. Values are Mean ± SE. Figure 7B - Conjugated bilirubin. Analysis shows an effect of: treatment P< 0.0087, time P< 0.0318, treatment-time interaction P< 0.0001, individual animal P< 0.0236. *P< 0.05, **P< 0.01, ***P< 0.0001. SEM = 0.192. Values are Mean ± SE. Bilirubin plasma levels correspond to the levels of free plasma hemoglobin. Therefore, the high levels observed for PG, and not for the glycerol treatment, suggest that at least part of the observed toxicity for PG (Figure 6) is due to hemolytic activity against red blood cells.

Detailed Description of Embodiments of the Invention

Sheep may be used as a model for studying human ketoacidosis. Pregnancy toxemia and lactation ketosis are major periparturient metabolic disorders of high-producing ruminants. During late pregnancy and/or early lactation, the energy demands peak and often exceed those consumed in the diet. The physiological adaptation to such a state of negative energy balance (NEB) is based primarily on adipose lipolysis and fat mobilization, which is transported in the circulation in the form of non-esterified fatty acids (NEFA) for hepatic catabolism to meet the physiological caloric demands. Excessive fat mobilization at glucose shortage enhances hepatic ketogenesis, thereby hyperketonemia and the risk for ketoacidosis.

Glycerol and propylene glycol (PG) are highly common cost-effective glucogens used to supplement the feed of ruminants. Both have been shown to reduce NEB associated with the transition period, and to help in diminishing pregnancy toxemia and ketosis. Metabolic effects of glycerol and PG have been studied by administration via drenching, as a feed-top dress or via ruminal infusion of cannulated animals, thus exposing them to substantial metabolism in the digestive system.

In order to compare their direct effect on NEB in sheep, with minimal interference from the digestive system, 5-months old ewe lambs induced for NEB by feed restriction, were intravenously (IV) treated with equicaloric doses of glycerol and PG, and blood parameters indicative of their energetic standing were monitored. The study showed clear distinctions between the metabolic impacts of glycerol and PG on NEB, which can be utilized in the development of new protocols for treatment of energy-deficient metabolic disorders, not only in ruminants but also in humans.

The inventors of the present application found that administration of PG and glycerol effectively reduced hyperketonemia by 57% and 61%, and inhibited adipose lipolysis by 73.6% and 73.3%, respectively. By contrast, glycerol was significantly more glucogenic than PG (glucose AUC of 3307 vs. 110 min x mg/dL, respectively; P< 0.0001), and more insulinotropic (insulin AUC of 14.4vs. -4.9 min x pg/L, respectively; P< 0.0075). Unlike glycerol, PG was substantially utilized for lactate formation (AUC of 82.4 vs. 228.4 min x mmol/L, respectively; P< 0.0002), with no apparent contribution to blood glucose formation. However, tissue-damage biomarkers indicated potential hemolytic activity for PG, which was not detected for glycerol.

The glycerol treatment may have reduced the hyperketonemia via several mechanisms likely contributing together. First, the substantial rise in glucose availability increases the abundance of the 4-carbon skeleton intermediates of the TCA cycle, affecting more directly oxaloacetate levels, thereby allowing more acetyl-CoA oxidation in the TCA cycle and its depletion as a precursor for ketone bodies synthesis. Secondly, the significant stimulation of plasma insulin by the glycerol treatment should signal directly for inhibition of ketogenesis through mTORCl inhibition of PPARcx. Based on the kinetics of the insulin and glucose responses to the glycerol treatment (Figures 2 and 4), the insulinotropic properties of glycerol appear secondary to the rise in blood glucose.

According to one aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of glycerol for use in treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis. ln some embodiments of the invention, the amount of glycerol in the pharmaceutical composition is between 5% and 22%.

The pharmaceutical composition comprising glycerol of the invention is used for treating ketoacidosis. According to some embodiments, the ketoacidosis is Diabetic ketoacidosis (DKA). In some other embodiments, the ketoacidosis is alcoholic ketoacidosis.

A further aspect of the invention relates to a method for treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis by administering a therapeutically effective amount of pharmaceutical composition comprising glycerol. In some embodiments, the ketoacidosis is Diabetic ketoacidosis (DKA). In some of these embodiments, the amount of glycerol in the composition is between 5% and 22%. In yet some further embodiments, the composition is administered intravenously.

As used herein, the terms "treatment", "prevention" and "prophylaxis" refer to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, ketoacidodsis. More specifically, treatment or prevention includes the prevention or postponement of development of the ketoacidodsis, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing additional symptoms and ameliorating or preventing the underlying causes of symptoms.

The term "treat" or "treatment", as used herein, refers to any type of treatment that imparts a benefit to a patient afflicted with a ketoacidodsis, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc. As used herein, to provide a "preventive treatment" or "prophylactic treatment" means protective treatment, to defend against or prevent something, especially a condition or disease.

As used herein, the term "ketoacidosis" as used herein refers to a metabolic state associated with high concentrations of ketone bodies, formed by the breakdown of fatty acids. In ketoacidosis, the body is unable to adequately regulate ketone production thereby leading to extremely high levels of keto acids in the blood to a point where the pH of the blood is substantially decreased. The term encompasses both diabetic ketoacidosis (DKA) and alcoholic ketoacidosis.

As used herein, "disease", "disorder", "condition" and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

As used herein, the term "pharmaceutical composition" refers to an active compound in any form suitable for effective administration to a subject, e.g., a mixture of the compound and one or more pharmaceutically acceptable additives.

As used herein, the term "pharmaceutically acceptable additive" refers to preservatives, antioxidants, fragrances, emulsifiers, dyes and excipients known or used in the field of drug formulation which do not interfere with the activity of glycerol, and are non-toxic.

As used herein, the term "excipients" is conventionally known to mean carriers, diluents and/or vehicles used in formulating drug compositions effective for the desired use.

As used herein, the term "therapeutically effective amount" is intended to mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, animal or human that is being sought by a researcher, veterinarian, medical doctor, dentist, periodontist or other clinician, or by the subject himself. In some embodiments of the invention, the pharmaceutical composition is administered intravenously (IV). As used herein, the term "intravenous (IV) administration" or "administered intravenously (IV)" relates to administration of the composition of the present invention by intravenous injection (preferably, by IV drip or IV infusion over a period of time).

In a preferred embodiment, compositions comprising glycerol are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent or other additives such as a local anesthetic.

Although embodiments of the invention have been described by way of illustration, it will be understood that the invention may be carried out with many variations, modifications, and adaptations, without exceeding the scope of the claims.

Examples

Example 1

Animal studies and experimental design

Sixteen ewe lambs of the Afec-Assaf breed (Gootwine, E., Reicher, S., Rozov, A., 2008 Prolificacy and lamb survival at birth in Awassi and Assaf sheep carrying the FecB (Booroola) mutation. Anim Reprod Sci 108, 402-411) (n = 8 per treatment; 4.5 ± 0.08 months old; 44.3 ± 0.85 kg in body weight) were randomly assigned to two treatment groups, while maintaining similar body weights distribution between the treatments. Typically, these lambs are weaned at 1.5 months and raised under intensive management (fed a ration containing 8 % oat hay, and 92% grain concentrate at 16% protein content) to maximize growth and weight gain (averaged at 300 gr/day). To induce NEB, the animals were fed wheat straw only ad libitum for 2 days, followed by another day of fasting and free access to fresh water. On the morning of the fourth day, at subclinical hyperketonemia and hypoglycemia levels, IV treatments were initiated with infusion of either glycerol or PG. Based on pilot experiments it was determined that the blood glucose and b-hydroxybutyrate (BHBA) return to baseline within roughly 5.5 hours of treatment, and accordingly blood sampling in this study was set for 6.3 hours. At the end of the experiment, the animals were returned to their normal diet gradually over a period of 3 days.

A single indwelling catheter (Delta Med SpA, Viadana, Italy) was installed on the jugular veins of all the animals a day before the experiment. To prevent coagulation, 3-6 mL of 10 lU/mL heparin (LEO Pharma A/S, Ballerup, Denmark) in sterile 0.9% saline (Teva Medical Ltd., Ashdod, Israel) were used to flush the catheter. Each sheep was infused with 170 mL of 0.22 pm filter-sterilized 15% glycerol (Bio-Lab Ltd, Jerusalem, Israel) or PG solutions (100% PG, Biovac, Or Akiva, Israel) in isotonic saline.

Blood samples of 5 mL were collected from each animal into heparinized vacutainers (Vacutainer; Becton Dickinson and Co., Franklin Lakes, NJ) immediately before treatments and within: 15, 40, 60, 140, 200, 270, 330, 380 minutes of the treatments. Blood glucose and BHBA concentrations were measured using the Freestyle Optium glucometer (Abbot Diabetes Care Ltd., Oxfordshire, UK). To harvest plasma, the heparinized blood was centrifuged at 2000xg for 15 minutes at 4°C. The supernatants (isolated plasma) were immediately stored at -20°C until further analysis.

Plasma biochemical analysis

Plasma lactate, Lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and bilirubin were analyzed using the Cobas C 111 analyzer (Roche Diagnostics, Rotkruez, Switzerland). Plasma ovine insulin was measured by ELISA (Mercodia, Uppsala, Sweden), and plasma NEFA were measured using a NEFA kit (Wako Chemicals, GmbH, Neuss, Germany).

Statistical analysis

Data of continuous dependent variables (glucose, BHBA, lactate, NEFA, Insulin, LDH, AST, ALT, total and conjugated bilirubin) were analyzed via the repeated measures ANOVA approach in JMP (VERSION 13.0.0, SAS Institute Inc., Cary, NC, 2016). The model included three fixed factors (Treatment, Time and Treatment X Time) and one random factor (Animal) nested within the Treatment variable. The Treatment had two levels (glycerol and PG), and the Sampling Time from treatment (time from injection) was treated as a nominal variable of 9 levels. Comparisons between the treatments at specific sampling times were carried out via contrast t-tests.

The area under the curve (AUC) was determined by the trapezoidal rule, computed as the total area between the response curve and the baseline. The baseline for each curve was obtained from a straight line connecting the first and the last measurement values. Differences between AUC and/or delta values (difference between the peak and the pretreatment values in percentage) were analyzed by Student's t-tests. Significance was accepted at P< 0.05. The effect of the animal body weights on their AUCs was not significant (P<0.419 as tested via standard least squares), therefore, they were not included in the final statistical model.

Example 2

Effect of glycerol and propylene glycol on energetic-state related factors

As a first step in studying the effects of IV treatments with glycerol and PG on the energetic status of sheep, all 16 ewe lambs were initially induced for NEB. This resulted in moderate but steady hyperketonemia, with an average BHBA concentration of 0.71 ±0.17 mM, and a relative hypoglycemia with average blood glucose values down to 68 % of their non-fasting basal values (56.7 ±5.9 vs. 82.7 ±6.1, respectively).

All the measured energetic-state related factors varied with the Sampling Time (P< 0.0034; Table 1), as expected from administration of single bolus of energy. Therefore, the responses of these factors were evaluated by the AUC of their concentration as a function of the sampling time, to obtain a more inclusive picture and statistically valid analyses of the treatments effects (Matthews, J.N.S., Altman, D.G., Campbell, M.J., Royston, P., 1990. Analysis of serial measurements in medical research. Br. Med. J.). The maximal magnitude of the response (delta) was found as another informative measure. Table 1: Effect of glycerol and propylene glycol (PG) intravenous infusion on circulating energy-homeostasis factors.

Delta % = (peak - pretreatment value)X100/(average pretreatment value),

Mean: least squares mean, SEM: standard error of the mean, NA: not applicable

*Statistical analysis refers only to the lasting time of the signal (first 4.5 hours).

Glycerol and propylene glycol were both anti-ketogenic, but only glycerol was glucogenic Following the IV treatments, the BHBA concentrations dropped markedly down by 61% (with glycerol) and 51% (with PG) of their initial subclinical levels (Figure 1). Notably, the response to PG was faster, reaching a minimum within 40 min of treatment compared to a minimum at 140 min from the glycerol treatment. However, the overall response had a more pronounced bell-shaped curve for glycerol (Figure 1). Despite the kinetic differences between the BHBA responses, the overall magnitude of the effect, which is better represented by the AUC, was not significantly different between the treatments (Table 1), indicating that both PG and glycerol are efficiently utilized to reduce ketogenesis.

Unlike their similar effects on hyperketonemia, the glycerol treatment induced a significantly greater glucose response than the PG treatment (Figure 2), as indicated by their respective AUC values (3306.7 vs. 110.4 min x mg/dL; P<0.0001, Table 1). Blood glucose levels increased immediately following the glycerol treatment and peaked within 1 hour, while in response to the PG treatment they remained stably around basal values (delta of 43.9% and 9.56%, respectively; P< 0.0006).

Unlike glycerol, propylene glycol raised circulating lactate

Mammals metabolize PG to lactate via hepatic alcohol and aldehyde dehydrogenases. The conversion of lactate to pyruvate can fuel both hepatic gluconeogenesis and oxidative metabolism. Moreover, like glucose, lactate can also directly support the fetal placental unit during pregnancy. In contrast to PG, glycerol can serve as a source for lactate only indirectly. Namely, after it enters gluconeogenesis, followed by the glycolytic pathway, which breaks down the resulting glucose to pyruvate, then to lactate via lactate dehydrogenase. However, at insufficient glucose balance, as in NEB physiology, this pathway is not expected to be significant since the rate of glycolysis is low. Consistently, the plasma lactate response was significantly lower with the glycerol treatment than with the PG treatment (AUC of 82.4 vs. 228.4 min x mmol/L, respectively, P< 0.0002; Table 1). Immediately following the PG treatment, lactate concentration increased sharply and peaked within 40 minutes, whereas with the glycerol treatment, lactate concentration remained relatively low throughout the sampling period (Figure 3).

Glycerol stimulates substantial plasma insulin compared to propylene glycol

Among the multitude of anabolic actions insulin induces to balance nutrient availability and demands, the stimulation of uptake of circulating carbohydrates at target tissues, and its ability to downregulate hepatic gluconeogenesis, establish this hormone as the master regulator of blood glucose levels. Insulin also downregulates adipose lipolysis and hepatic ketogenesis. Therefore, to better understand the effects of the glucogenic treatments on hypoglycemia and hyperketonemia, we monitored their simultaneous effects on circulating insulin levels. The glycerol treatment led to a significantly greater insulin response than the PG treatment (AUC of 14.4 vs. -4.91 min x pg/L, respectively; P< 0.0075, Table 1). Insulin concentration peaked 2 hours following the glycerol treatment, yet it did not increase significantly above baseline with the PG treatment (Figure 4).

Example 3

Both glycerol and propylene glycol are anti-lipolvtic. but the response to glycerol is faster

The primary mechanism, by which stored energy in adipose tissue is delivered to calories demanding tissues, is mediated via fat mobilization in the form of NEFA. Therefore, increase in circulating NEFA serves as the major plasma indicator of adipose lipolysis, as well as a key feature of the NEB physiology, in addition to hypoglycemia and hyperketonemia. Both glycerol and PG significantly decreased the plasma NEFA concentrations, which dropped within 3.5 hours down to 73% and 64% of the baseline values, respectively (Figure 5). However, the NEFA response to the glycerol treatment was much faster; with a substantial drop within 60 min of treatment compared to a substantial drop only 200 min after the PG treatment. Overall, the glycerol treatment was initially more significant up to 140 min from treatments, afterward the NEFA values were similarly low for both treatments.

Example 4

Unlike glycerol, propylene glycol may have induced tissue damage and hemolysis

LDH, ALT and AST exist in the liver and in other tissues such as heart and muscle. In case of tissue damage their plasma levels increase and therefore they are commonly used as biomarkers of liver and tissue damage. PG increased the concentration of LDH, ALT and of AST 40 minutes following treatment when compared with glycerol (Table 2; Figure 6). Plasma samples isolated from sheep treated with PG had significant reddish coloration compared to those of the glycerol treated animals, which had a normal yellowish color. Consistent with this strong indication for hemolytic activity, the plasma bilirubin levels also increased significantly with the PG compared to the glycerol treatment (Figure 7 and Table 2).

Table 2: Liver and tissue damage biomarkers in response to glycerol and propylene glycol (PG) intravenous infusion.

Delta % = (peak - pretreatment value)X100/(average pretreatment value), Mean: least squares mean, SEM: standard error of the mean, NA: not applicable

The significant increase in liver health biomarkers following treatment with PG, but not with glycerol, indicates that PG has potential adverse effects. Taken together with the hemolytic plasma coloration with the PG treatment, and the rise of bilirubin (Figure 7) which is highly related to the free circulating hemoglobin, it is plausible that the potentially toxic activity of PG arises from damage to red blood cells. Accordingly, glycerol IV administration stems as a safe (non-toxic) and efficient treatment for ketoacidosis. Although embodiments of the invention have been described by way of illustration, it will be understood that the invention may be carried out with many variations, modifications, and adaptations, without exceeding the scope of the claims.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of glycerol for use in treating, preventing, ameliorating, reducing or delaying the onset of ketoacidosis.

2. The pharmaceutical composition for use according to claim 1, wherein the amount of glycerol in the composition is between 5% and 22%.

3. The pharmaceutical composition for use according to claim 1, wherein said ketoacidosis is Diabetic ketoacidosis (DKA).

4. The pharmaceutical composition for use according to claim 1, wherein said composition is administered intravenously.

5. The pharmaceutical composition for use according to claim 1, wherein said composition is administered simultaneously with SGLTi (sodium-glucose linked transporters inhibitors).