DOI: 10.31038/EDMJ.2019323

Abstract

It has generally been assumed that hormones and the corresponding intra- and intercellular signal transduction pathways and mechanisms have evolved exclusively during course of the evolution of vertebrate endocrine organs, implying a rather recent origin. However, there is good experimental evidence for (i) the expression of hormones and hormone-binding proteins resembling those of vertebrates in fungi and yeast, (ii) functional responses of lower eukaryotes to mammalian hormones and (iii) the existence of components of insulin-like and mimetic signaling pathways as well as their coupling to G-protein coupled receptors and metabolic pathways, such as lipolysis and endoplasmic reticulum stress, in lower eukaryotes, in particular in Neurospora crassa and Saccharomyces cerevisiae. Data will be presented that the naturally occurring or recombinant expression of insulin-like/mimetic signaling pathways in lower eukaryotic cells may be useful as model systems for future drug screening and discovery efforts.

Keywords

Diabetes; Drug Screening; Endoplasmic Reticulum Stress; Glucagon-like Peptides; G-protein-coupled Receptors; Insulin; Lipolysis, Saccharomyces cerevisiae

1. Introduction: Models for the discovery of insulin-mimetic and anti-diabetic drugs

The yeast Saccharomyces cerevisiae, as a simple and well-understood eukaryotic model organism, offers direct means to unequivocally assess processes that couple ligand binding to receptor activation, formation of transient intermediates, and downstream signaling to the molecular targets of the activated receptor. For example, events proximal to insulin-binding at the receptor can be traced to distal events to up to the terminal effector systems, such as dephosphorylation of GS or phosphorylation of ribosomal proteins. Furthermore, the interactions between multiple mutations, their suppressors, and ultimately, between the molecular participants, in total, in an insulin-activated response cascade can be readily tested in yeast. The ease of biochemical, cell biological, genetic and molecular biological manipulation has made S. cerevisiae an excellent model for the study of metabolic control in eukaryotic cells. In fact, yeast can be considered as the preferred model eukaryotic organism for the following reasons: I) Many cellular processes are conserved between yeast and higher eukaryotes, including (i) metabolic and energy-generating pathways, (ii) signal transduction pathways, (iii) protein targeting to specific subcellular locations, e.g. secretion, biogenesis of plasma membranes and mitochondria, (iv) peptide maturation, e.g. signal sequence cleavage, prohormone cleavage at Lys-Arg residues, (v) protein modification, e.g. Ser/Thr/Tyr phosphorylation, N- and O-glycosylation, acylation, attachment of GPI anchors, (vi) nuclear processes, e.g. transcription, RNA processing and polyadenylation. II) Many proteins of yeast are very close orthologs to mammalian counterparts, and their functional replacement by the latter has been shown for some yeast components (Table 1), signal transduction components such as protein kinases and phosphatases (Table 2). III) Yeast represents the best understood eukaryotic organism at the genetic level under the control of a compact and simple genome with few introns, repetitive sequences and non-coding elements. IV) Yeast is ideally suited for both classical and molecular genetic manipulation with many powerful tools introduced during the past five decades, including (i) high and low copy number plasmid vectors, (ii) strong and inducible promoters, (iii) high transformation efficiency (> 10⁵/µg of DNA), (iv) gene replacement in one week, (v) identification and cloning of genes acting along a pathway with the methods of functional complementation, genetic suppression, synthetic lethality etc.

Table 1. Structural comparison of some signal transduction proteins/genes between mammals and yeast with regard to %identity of amino acids. As indicated some mammalian genes can restore the function of the disrupted homologous gene in Saccharomyces cerevisiae.

Table 2. Some selected yeast homologs of mammalian protein kinases and phosphatases with established function in signal transduction.

2. G-Protein Coupled Receptors as Targets

G-protein-coupled receptors (GPCRs) as transmembrane proteins represent components of intracellular signaling cascades which transduce information from the cell surface into the cell interior in course of agonist binding and the resulting activation of heterotrimeric G-proteins. GPCRs together with their corresponding elements of downstream signaling have been found to operate in all eukaryotes from yeast to mammals [1] and to share pronounced sequence similarities between yeast and mammals (Table 3). It has recently been recognized that in mammalian cells a complex interplay and mutual cross-talk exists between the insulin signaling cascade and the signaling pathways directed by GPCRs. It depends on the type of GPCR whether insulin action is modulated in a positive or negative fashion and thus insulin sensitivity becomes increased or diminished (i.e. insulin sensitizing or desensitizing) [2–8]. The basis of the successful screening for anti-diabetic drugs is formed by modeling of the activation of the G-protein-coupled class of human receptors in yeast. The successful coupling of human receptor activation to the mating pathway of yeast has been demonstrated [9–11] which should thereby enable the screening for both agonists and antagonists, depending on the needs. The putative targets encompass known members of the GPCR class which are assumed to be involved in the pathogenesis of T2D, among them the glucagon receptor, glucagon-like-peptide 1 receptor (GLP-1) and a variety of α- and β-adrenergic receptor subtypes, such as muscarinic acetylcholine receptors and various receptors of the brain and nervous system. Significant applications to many other therapeutic areas can be expected where GPCR members play a role.

Table 3. Some selected yeast homologs of mammalian cAMP and G-protein signaling components, acylation factors, and lipid kinases with established function in signal transduction.

The experimental procedure involves (i) targeted mutagenesis of the endogenous GPCR and G_α genes, (ii) covalent linkage of the human GPCR to yeast G_α by gene fusion to overcome the lack of recognition specificity between the human GPCR and the yeast G_α, and (iii) mutagenesis of the G_α domain and selection for functional coupling to ensure efficient interaction (Figure 1). The functional assays designed to enable convenient measurements and clear-cut read-outs are typically, but not exclusively, based on (i) growth arrest and morphological change to “shmoos”, (ii) auxotrophic selection, i.e. activation of the GPCR leads to expression of an essential gene which will support growth in minimal medium, (iii) color development, i.e. activation of the GPCR leads to expression of β-galactosidase, (iv) fluorescence, i.e. activation of the GPCR leads to expression of A. victoria green fluorescent protein (Figure 2).

Figure 1. Two different modes of activation of heterotrimeric G-proteins by receptors are feasible in yeast cells. In the unlinked physiological mode of interaction between receptor and G-protein, the activation depends on the affinity between both partners (left section). A possible “mismatch” between a mammalian receptor expressed in yeast and the endogenous G-protein may be overruled by covalent linkage to G_α to the receptor. Nevertheless, the engineered covalent hybrid receptor-G-protein signaling complex in yeast is responsive towards activation/dissociation of the endogenous G_β/G_γ-subunits by ligand binding to the mammalian receptor “subdomain” (right section).

Figure 2. Engineering yeast for drug discovery requiries functional reconstitution of transmembrane signaling (1) which can be coupled to a signaling pathway regulating growth (2) or transcription of a reporter gene (e.g. ß-galactosidase) enabling a simple color assay (3).

Figure 3. A multitude of orthologous components is engaged in signaling via heterotrimeric G-proteins in yeast (right section) and mammals (left section)

2.1. General Strategy

In yeast the heptahelical GPCR, Ste2, is engaged in intracellular signal transduction initiated by the α-factor pheromone (Figure 3) [12, 13]. Compatible with the evolutionarily conserved signaling function of Ste2, its expression in human HEK293 cells led to stimulation of the MAPK Erk1/2 upon incubation with α-factor [14]. Stimulation of Ste2 in yeast led to its endocytosis, ubiquitination and finally degradation, whereas in HEK293 cells, the α-factor-induced internalization of Ste2 was not accompanied by significant downregulation of the cellular amount of Ste2. The lack of expression of typical receptor tyrosine kinases, such as the insulin receptor and IGF-1 receptor, in yeast makes Ste2 a perfect target for the identification of the protein motifs which enable the regulation of GPCRs through mammalian tyrosine kinases and vice versa for the impact of GPCR expression and activation on receptor tyrosine kinase signaling, such as desensitization of the insulin receptor as the molecular mechanism for insulin resistance and T2D. For instance, in the basal state a Ste2-GFP fusion protein becomes targeted to the plasma membranes, but does not undergo endocytosis upon challenge of the yeast cells with insulin, as is typical for the GPCR ß₂-adrenergic receptor in course of agonist binding [14]. The failure of insulin to control the intracellular trafficking of Ste2 enabled the construction of a model system consisting of yeast Ste2 and mammalian cells for studying the protein motifs which are responsible for the “linear” or “single-hit” GPCR receptor biology (i.e. agonist binding > receptor activation > endocytosis > ubiquitination > proteasomal degradation) induced by typical GPCR ligands, such as ß-adrenergic agonists. In higher eukaryotic cells, such as insulin target cells (liver, muscle, adipose), the resensitization and recycling of endocytosed GPCRs is typical, which raises the question about the molecular mechanisms controlling the recycling efficacy and thereby the periods of cell-surface retention. Yeast GPCRs are known to operate independent of cross-talk from/to receptor tyrosine kinases, which could influence the cell surface expression of GPCRs in mammalian cells. However, in yeast the endocytosis of GPCRs is not coupled to their rapid degradation or recycling back to the cell surface. The molecular mode of the control of GPCR trafficking by receptor tyrosine kinases, such as the insulin receptor, turned out to encompass the direct tyrosine phosphorylation of the GPCR as well as the serine/threonine phosphorylation by downstream protein kinases, such as PKB/Akt. Thus the identification of the complete panel of amino acid motifs, phosphorylation sites and downstream binding events with adaptor proteins, such as Grb2, can be achieved by the recombinant introduction of selected regions of the mammalian GPCR of interest into yeast Ste2 and subsequent assaying for the fate of this chimeric receptor in response to the corresponding agonist in animal cells. Thereby it was previously shown that the downregulation by degradation of a chimeric yeast Ste2 upon challenge with insulin is conferred by substitution of the endogenous cytoplasmic domain of Ste2 with that of the ß₂-adrenergic receptor [14]. This model system will enable the screening for protein kinases, protein phosphatases and adaptor molecules which are engaged and required in the recycling/resensitization of a mammalian GPCR, such as ß₂-adrenergic receptor, under the control of a mammalian tyrosine kinase, such as the insulin receptor, and vice versa, of a tyrosine kinase under the control of a GPCR. Thus, the investigation of such endocytosed Ste2 chimera may be useful for the elucidation of novel drug targets as well as insulin-mimetic and insulin-sensitizing agents for the therapy of diabetes.

2.2. GLP-1 Receptor as Target

The finding that a more pronounced insulin secretion results from the administration of glucose via the oral compared to the intraveneous route prompted the postulation of the action of so-called incretins [15]. GLP-1 is a members of the incretins which is secreted by intestinal cells in response to nutrient ingestion. GLP-1 contributes to the regulation of blood glucose predominantly via the induction of insulin release from pancreatic ß-cells [16]. GLP-1 triggers this insulin-releasing effect by binding to and activation of a typical GPCR, the GLP-1 receptor [17]. Thereby, the GLP-1 receptor plays an important role in glucose-dependent insulin release and has gained major interest as a target for the identification of novel (insulin-releasing) drugs for the therapy of T2D [18]. Interestingly, T2D is not only characterized by reduced insulin sensitivity and capacity of insulin secretion, but by decreased serum concentrations of GLP-1, in addition [19]. Strikingly, the intravenous injection of GLP-1 peptide causes potent upregulation of glucose-dependent insulin secretion and counteracts hyperglycemia in T2D patients [20]. Unfortunately, the plasma half-life of GLP-1 (7–36 amide) as the active version of GLP-1 is very short due to rapid cleavage to the inactive version GLP-1 (9–36 amide) by the serine protease dipeptidyl peptidase IV [21]. To overcome this limitation and to improve plasma half-life, a number of DPP-IV-resistant peptidic GLP-1 analogues, such as exenatide and liraglutide (Table 4), were developed which meanwhile have been introduced into the therapy of T2D [22].

In fact, these GLP-1 mimetics turned out to have important advantages in the control of T2D, such as rapid weight loss and low risk for hypoglycemic episodes, but their chronic application has been implicated with elevated risk of cancer and pancreatitis [23]. Unexpectedly, different GLP-1 mimetics sharing pronounced sequence homology exhibit considerable differences in their clinical profile [24]. This heterogeneity may be explained by the engagement of multiple isoforms of the GLP-1 receptor or, alternatively, the differential activation of the GLP-1 receptor with regard to its ability to coupling to and activate distinct G-proteins and thereby to induce distinct downsteam signaling pathways. Thus, much remains to be learnt about the biology of the GLP-1 receptor, in particular the (different) mode(s) of interaction of ligands and the resulting differential consequence(s) for the downstream responses. This inadequate and incomplete understanding of the molecular mechanisms of GLP-1 action in combination with the concerns regarding the safety of GLP-1 mimetics led to their categorization as “tier 2” of the consensus algorithm for initiation and adjustment therapy for the American Diabetes Association and European Association for the Study of Diabetes. Under this category the “less well-validated” treatments are encompassed and recommened as the last-line treatment of patients. This judgement can only be revised on the basis of a better knowledge of the differential activation and downstream signaling of the GLP-1 receptor in course of ligand binding. Corrresponding studies should facilitate the development of GLP-1 mimetic drugs with improved efficacy and safety.

The conventional assays used so far for studying GLP-1 receptor signaling are affected by other signaling pathways which manage to cross-talk to the receptor. Recently a robust and simple yeast-based assay system has been introduced which enables the monitoring of single GPCR-G-protein couplings with the aim to decipher the impact of a defined G-protein subunit on GLP-1 receptor downstream signaling [26]. Previously established test systems for the measurement of the dissociation constants of agonists and antagonists as well as relative binding efficacy of agonists have demonstrated the robustness of signaling assays using Saccharomyces cerevisiae cells [27]. Even more importantly, since the GPCR-G-protein interaction is defined in unequivocal fashion, the putative bias of a given (expressed) GPCR or G-protein for the interaction with a certain G-protein or GPCR, respectively, and corresponding consequences for downstream signaling can be elucidated rather conveniently without confounding effects potentially caused by other GPCRs and competing G-proteins. Subsequently, this yeast signaling assay has been adapted to establish the G-protein bias profiles for a number of GLP-1 mimetics (Table 4) [28].

Table 4. Comparison of GLP-1 receptor peptide ligands and relative bias factors (adapted from and modified according to Weston 2014). The sequences of the various peptide ligands for the GLP-1 receptor are aligned to the natural agonist (GLP-1). Deviations in amino acid sequence from GLP-1 of the other GPCR ligands are highlighted in blue. The relative (to GLP-1) bias factor was quantitatively evaluated for each ligand as the change in log(τ/K_A) ratio where a negative value indicates preference for the inhibitory G_αi chimera. Statistical significance was determined using one-way ANOVA with Bonferroni’s post-test with each data set compared with GLP-1 (**P < 0.01, ***P < 0.001). Data are mean of 5–8 independent experiments + SEM.

The use of yeast compared to other (e.g. mammalian) test systems has the benefit of providing a relatively zero background for G-protein activation which allows the identification of activation profiles at the individual level. This is of particular importance for the GLP-1 receptor since it is known that this receptor like other GPCRs is known to couple to a multitude of different G-proteins. The predominant one is the G_αs subunit which upon activation signals for the stimulation of cAMP generation. A minor one is the pertussis toxin-sensitive inhibitory G_αi [29, 30], the functional outcome of this coupling however remains to be characterized in greater detail [31]. In fact, the productive coupling of GLP-1 to G_αi was confirmed [28] with the use of the yeast chimeric G_α system [26, 32, 33]. GLP-1 as the natural peptide ligand elicited the concentration-dependent induction of the GPA1/G_α1 chimera with significantly diminished potency (EC₅₀) and efficacy (log τ) in comparison to signaling through GPA1/G_αs. Interestingly, antagonism of the signaling event was left unaltered. These findings can be interpreted with the G-protein subunit expressed being irrelevant for the antagonist affinity and the measured alterations in agonist affinity being due to the preference for a certain G-protein, but independent of the use of the yeast assay system. Significant differences between the two systems were not measured with regard to the dissociation constant for the receptor antagonist, exendin-3. In contrast, for the therapeutically relevant GLP-1 ligands, liraglutide and exenatide, considerable but previously unrecognized differences in G-protein signaling were detected with exenatide exhibiting a pronounced bias for the G_αi pathway. Subsequently, this approach was extended to the investigation of small-molecule allosteric compounds and the closely related GPCR glucagon receptor. In conclusion, the yeast GPCR assay system allows the reliable and system-independent measurement of the pharmacological properties of putative GPCR-based drugs. The glucagon receptor as well as the GLP-1 receptor belong to the secretin (family B, class 2) GPCR family with 15 members. The peptidic ligands for those receptors typically consist of 27 to 84 amino acids.

2.3. The Use of Permeable Yeast Cells

For decades the investigation of GPCRs in yeast was not favored by the researchers since the yeast cell wall was thought to prevent their naturally occurring peptidic ligands from reaching the (periplasmic face of the outer) plasma membranes, where the ligand binding domain of the (ectopically) expressed GPCRs will be located. However, recently the ectopic expression of two corticotropin-releasing factor receptor subtypes [34, 35] as well as the functional co-expression of the calcitonin receptor-like receptor together with a variety of distinct receptor activity-modifying proteins (RAMPs) [36] has been demonstrated. In apparent conflict with the above assumption of the non-permeation of GPCR ligands across the yeast cell wall, in those cases as is true for the glucagon receptor and the GLP-1 receptor, the yeast-based assay system succeeded in the accurate reproduction of the in vitro pharmacology (e.g. relative binding affinities, G-protein coupling), albeit with absolute binding affinities typically below those measured in corresponding mammalian test systems. Nevertheless or to overcome the latter limitation permeable yeast cells have been developed.

Microbial cells have evolved an impermeable cell wall and plasma membranes that allow them to survive in the environment. Therefore the drug target may not easily accessible to compounds in high-throughput screens that use microbial cells, which in consequence are likely to miss those compounds being unable to penetrate across the cell wall and plasma membrane barriers. In addition, many microbes have very effective efflux systems that pump out compounds. These efflux systems are similar to the multidrug resistance (MDR) transporters found in tumor cells. One of the large classes of efflux systems, or transporter, is called the ATP-binding cassette transporters or ABC transporters. The ABC transporters are conserved fom bacteria to man [37]. With such drawbacks, can microbial- and yeast-based screening be effectively used for drug discovery? Genetic and molecular technology has made it possible to remove some of these barriers and make screen development and screening in unicellular lower eukaryotes a viable, inexpensive, and productive alternative to other screening systems. Among lower eukaryotes, Saccharomyces cerevisiae have been the most popular because of the genetic manipulations feasible with this organism. Since it is an eukaryote, it is often considered to be a more realistic system for screening for mammalian drug targets as compared to prokaryotes, such as E. coli. However, yeast is slower to grow than E. coli, taking 48 h to grow to adequate cell densities, while E. coli can be used within 6 to 8 h of growth. The genetic manipulations in E. coli are considerably less difficult than with yeast and E. coli are more permeable than yeast.

In fact, wild-type Saccharomyces cerevisiae is quite impermeable owing to their cell wall and plasma membranes. The cell wall is considered to be latticelike and allows most small molecules to permeate through. However, the plasma membranes are considered to be quite impermeable. Strikingly, previously it has been noted that yeast cells are actually permeable and the lack of drug effect is the result of the activity of multiple efflux systems, belonging to the family of ATP-binding cassette transporters (MDR), called PDR, that rapidly pump out compounds. The transcription factors, pdr1p and pdr3p, down-regulate the expression of hexose transporters, HXT11 and HXT9, which in turn up-regulate the expression of PDR (Figure 4). Thus deleting the hexose transporters, HXT11 or HXT9, confers pleiotropic drug resistance on yeast while overexpression of these transporters results in increased sensitivity to drugs. Furthermore, deletion of the regulators of the promoter for the ATP-binding transporters, PDR1 and PDR3, in HXT11 and HXT9 over-expressing strains, results in supersensitive yeast [38]. These mutant strains are ideal organisms for use as host strains for the development of screens. Improved cell permeability was also reported for the yeast strains with deletion of the YOR1 gene, which encodes another ATP-binding cassette transport  protein [39]. In addition to transporter mutants, mutants in the ergosterol pathway, such as in ERG6, are also more permeable to small molecules used in screening compared to wild-type strains [40]. However, the mutants in the ergosterol pathway have the disadvantage that their growth is negatively affected, and they are difficult to transform.

Figure 4. Insulin signal transduction may regulate the MAPK cascade in yeast (right section). Insulin signaling in insulin-responsive mammalian cells via the homologous Ras-MAPK pathway is shown for comparison (left section). In yeast coupling of the endogenous (ScIRP) or heterologously expressed human (hIR) insulin receptor to the MAPK pathway could occur via the heterotrimeric G-protein, GPA1/STE4/STE18 to STE20 (which is also used by the pheromone receptor during the mating response) or via CDC25 to Ras1/2, respectively, which may be analogous to mammalian cells where the insulin receptor transduces its signal to Ras via the GDP-GTP exchange factor SOS.

It would be useful and feasible to extend this technology for the inclusion of many GPCRs or other components, which putatively interact with the receptor in situ (e.g. regulatory proteins of G-protein signaling, RAMPs), in order to recognize any (positive or negative) effect of an interaction built up on the receptor pharmacology and the resulting (patho)physiological changes. Moreover, the relative simplicity and convenience of the ectopic expression of GPCRs in yeast would be compatible with the introduction of patient-specific mutations into GPCRs with the aim to study and to quantitatively analyze their impact on the ligand-induced signaling capability of the receptor and possibly to efficiently screen for ligand mimetics, which bypass the defect. Thereby, the yeast-based assay system could contribute to the development of personalized drugs.

3. Insulin Receptor and Signaling as Target

This approach originated during an attempt to study the molecular basis for tyrosine kinase-linked signal transduction in a cell system in which there is no detectable endogenous receptor tyrosine kinase-coupled regulatory processes, namely, the fission yeast Schizosaccharomyces pombe. Expression of selected mammalian signaling elements in such a naive system might support productive interactions between heterologous components while eliminating involvement and cross-talk with host cell tyrosine kinase-associated control systems. S. pombe has been used by others to study the function and interactions of selected mammalian signaling elements, including PKC isoforms, Src, Csk, Raf-1, Ras, PI-3’K and MAPKK. Furthermore, high level expression of the platelet-derived growth factor receptor and its substrate phospholipase Cγ (PLCγ) in S. pombe led to autophosphoryation and substrate phosphorylation at tyrosine residues. Therefore, appropriate cleavage and oligomerization of the expressed human receptor seem to be possible. Potential assays may rely on (i) modulation of growth, (ii) insulin binding, (iii) autophosphorylation of the receptor at tyrosine residues, (iv) tyrosine phosphorylation of heterogenously expressed authentic or engineered substrates, such as IRS-1 and PI3K as well as fusion proteins or fragments derived thereof, or artificial substrates, e.g. EEEY and (v) phosphorylation of yeast substrates (Figure 4). These experimental designs should demonstrate, that heterologous expression of selected signaling elements in yeast will permit the detection and analysis of functional insulin receptor activation and pave the way for further systematic and detailed molecular dissection of a wide range of interactions, central to insulin receptor kinase-coupled signal transduction processes as well as for the identification of peptidic or small peptidomimetic molecules, which manage to strengthen or disrupt those interactions.

Alternatively, the assays for human receptor activation may be based on signal transduction pathways activated by receptor tyrosine kinase activity: I. The soluble domain of the human insulin receptor can be stably expressed at the plasma membranes of Saccharomyces cerevisiae [42]. II.Tyrosine phosphorylation of some yeast proteins is increased upon incubation of intact cells or spheroblasts with human insulin [43]. This raises the possibility that the tyrosine kinase activity of the human insulin receptor may initiate signal transduction cascades leading to phosphorylation of endogenous substrates, and their human orthologs expressed in yeast. This will require (i) cloning of the yeast signaling proteins that respond to the human insulin and insulin-dependent (tyrosine/serine/threonine) phosphorylation, (ii) identification and (iii) expression of their human orthologs. III. Low levels of expression of the insulin receptor kinase domain may lead to a very modest but reproducible slowing of growth, high expression may result in stronger inhibition as has been reported previously for overexpression of the constitutively active human insulin receptor in cultured CHO cells [44]. In general, clear-cut effects of activated yeast or human receptors on downstream signaling proteins of yeast or human origin represent potential parameters of measurement for a yeast-based screening assay. Taken together, yeast and other lower eukaryotic unicellular organisms have already proven their usefulness in various aspects of pharmaceutical development and, no doubt, their impact will further increase during the next years.

4. Endoplasmic Reticulum Stress and Pancreatic ß-Cell Dysfunction as Target

Failure of insulin secretion caused by reduction in the number and functionality of pancreatic ß-cells has been considered as the hallmark of T2D for decades [45, 46]. The majority of T2D patients is characterized by overweight or obesity which in general is correlated to elevated levels of plasma free fatty acid (FFA) levels [47, 48]. Albeit being still a matter of intense debate, this hyperlipidemic state could be (causally) involved in the loss of functional ß-cells through a pathophysiological process called lipotoxicity [49]. Importantly, the operation of lipotoxic processes under conditions of overfeeding and excessive calorie uptake, as often prevalent during obesity and T2D, has also been reported for a number of non-ß-cells, such as adipocytes, myocytes, hepatocytes and brain cells, and linked to their desensitization towards insulin challenge [50–53]. The molecular mechanisms underlying the FFA-induced lipotoxicity have been investigated in vitro during the past two decades in course of direct incubation of cultured ß-cells, pancreatic islets or insulin target cells and, more recently, of yeast cells (Saccharomyces cerevisiae) [54–56]. Strikingly, despite the tremendous differences between these cell types considering their morphology, structure and physiology, they apparently display pronounced similarity with regard to their susceptibility and viability towards FFA (Table 5). This argues for conservation, at least in part, of lipotoxic mechanisms along the evolution of eukaryotes from yeast to humans. Importantly, in ß-cells, insulin target cells and yeast cells the extent of the lipotoxic effects provoked by the FFA critically depends on the number of their carbon atoms and the degree of their saturation. The data available so far are consistent with saturated (SFA) and long-chain FFA (e.g. palmitate C16:0, stearate C18:0) exerting the most adverse effects, whereas saturated and short(er)-chain ones (e.g. myristate C14:0 and below) as well as long-chain unsaturated fatty acids (UFA) equipped with one to several double bonds (e.g. palmitoleate C16:1, oleate C18:1, linoleate C18:2 and linolenate C18:3) exhibiting considerably lower harmfulness [57–59]. In addition, the concentration-dependent abrogation of the SFA-triggered lipotoxic effects, in general, and of cell death, in particular, by excess of UFA has meanwhile been amply documented [60, 61].

Most importantly, in both ß-cells and yeast cells the SFA-triggered lipotoxicity leads to the initiation of the so-called stress response in the endoplasmic reticulum (ER) or unfolded protein response (UPR)(Table 5). The ER represents the site for a number of essential physiological processes, such as Ca²⁺-homeostasis, (phospho)lipid biosynthesis and biogenesis of proteins, which have to be transported to intracellular organelles and the plasma membranes or secreted into the extracellular milieu. Prior to guidance into the secretory pathway the membrane and secretory protein precursors have to be correctly folded and assembled in the ER. Any imbalance between the folding capacity of the ER and its passenger protein load will ultimately cause ER stress [62]. ER stress has to be counterbalanced by the multiple strategies of the UPR, such as proteolytic degradation [63] to preserve or re-gain the structural and functional integrity of the ER despite the accumulation of misfolded polypeptides [64, 65]. It is believed that the SFA-triggered cell death is mediated through ER stress and the failure to cope with it in the presence of high concentrations of SFA, which finally will result in apoptosis.

Table 5. The effects of exposure of yeast- and ß-cells towards palmitate in comparison.

In higher eukaryotes, a critical pathway engaged by the UPR, which senses for unfolded membrane protein precursors, is constituted by the inositol-requiring enzyme 1 (IRE1) cascade [65], which in ß-cells seems to be upregulated in response to long-chain SFA, such as palmitate. The IRE1 cascade constitutes the only UPR pathway which is conserved from yeast to humans. In Saccharomyces cerevisiae the accumulation of misfolded proteins triggers dimer formation of Ire1p which results in upregulation of its endogenous endoribonuclease activity. In non-stressed yeast and ß-cells the ER-resident protein of the HSP70 family, Kar1p and BiP, respectively, binds to Ire1(p) for suppression of its activation. In stressed cells harboring excess of misfolded proteins in the ER Kar1p / BiP manages to dissociate from Ire1(p) and thereby enables Ire1(p) dimerization which finally leads to its autophosphorylation and activation [64]. The Ire1p endoribonuclease activity is directed against the mRNA of the transcription factor Hac1p which interacts with the promoter of UPR elements (UPREs) and controls the expression of about 5% of the genes in yeast (and is the yeast ortholog of mammalian XBP1). Importantly, in yeast upregulation of the Ire1p cascade in the presence of high concentrations of SFA was observed using a reporter gene (LACZ) assay which monitored the transcription activity of four UPREs, i.e. the amounts of active Hac1p/XBP1, as reflected in the measured ß-galactosidase activity [66]. High intracellular concentrations of SFA in yeast as well as in ß-cells, which may result either from blockade of endogenous fatty acid desaturation or from the presence of palmitate in the incubation medium, have been reported to provoke full induction of the Ire1(p) cascade. Consistent with the beneficial effects of UFA, Ire1(p) activation was diminished in concentration-dependent fashion in the presence of oleate [67].

5. Adipocyte Lipolysis as Target

The concentration of FFA in the blood of mammals which are tightly linked to their metabolic disease state, such as T2D in case of chronically elevated levels, critically depends on the efficacy of lipolysis in the various adipose tissue depots [68]. Insulin with its primarily anabolic function in the body manages to restrict lipolysis and to foster the incorporation of FFA, which are ultimately derived from the ingested nutrient lipids, into neutral triacylglycerol, which becomes stored in adipocytes, during the postprandial state. Missing suppression of adipocyte lipolysis by insulin has been considered since decades as the major physiological defect which is causative for metabolic diseases, such as T2D and obesity, through the induction of insulin resistance in peripheral tissues and dysfunction and death of ß-cells through the operation of lipotoxic mechanisms (see above) [69–71].

Triacylglycerol is lipolytically degraded to FFA and glycerol through the joint action of tri-, di- and monoacylglycerol lipases [72–75] with the most recently unraveled Adipose Triglyceride Lipase (ATGL) [76–78] exerting the major portion of the triacylglycerol hydrolase activity in adipose, muscle and liver tissues and representing the rate-limiting enzyme for lipolysis. This is manifested in the observation that up- and downregulation of ATGL expression unequivocally leads to increase and decrease, respectively, of both the basal and the cAMP-induced triacylglycerol hydrolysis [79–81]. It has been accepted for decades that the rate of lipolysis is primarily under the short-term and post-translational control of the cAMP-dependent signaling pathway through phosphorylation of the lipid droplet coat protein perilipin and the diacylglycerol lipase HSL by PKA leading to rapid stimulation of lipolysis, whereas insulin-induced blockade of cAMP-dependent signaling via Akt-dependent [82] and -independent [83] mechanisms causes inhibition of lipolysis.

In addition, insulin and other physiological anti-lipolytic stimuli, such as feeding and α-adrenergic hormones, have to elicit a long-term inhibitory effect on lipolysis which is based on the suppression of its rate-limiting enzyme, ATGL, most likely as a consequence of downregulation of ATGL expression. Recently, a novel molecular mechanism for the negative regulation of ATGL expression by insulin and nutrients has been identified, the mTORC1-dependent pathway which blocks lipolysis by reduction of ATGL transcription [84]. Interestingly, in Drosophila a similar mechanistic link between dTORC1 and the ATGL homologue Brummer lipase [79, 85] was demonstrated recently [86, 87], which even can be extended to the insulin receptor [88]. The apparent evolutionary conservation of this anti-lipolytic molecular mechanism strongly argues for its essential physiological role in eukaryotes, in general, and in humans, in particular, as well as makes it to an attractive source for targets for the therapy of metabolic diseases.

For elucidation of the molecular mode of mTORC1 action, yeast as a model organism was used based on the knowledge, that Saccharomyces cerevisiae harbors a functional ortholog of ATGL, the triglyceride lipase Tgl4p (Table 1) [80]. The putative involvement of the Tor1-dependent pathway in the expression of Tgl4 was studied by growing S. cerevisiae in the presence of the specific Tor1/mTORC1 inhibitor, rapamycin [81]. In fact, rapamycin led to a considerable and specific upregulation of the Tgl4p mRNA expression. For identification of the transcription factors engaged in Tgl4 expression, a S. cerevisiae deletion library was screened [81]. Interestingly, yeast cells lacking Msn4 were found to have increased basal levels of Tgl4 mRNA and to be almost insensitive towards the stimulatory effect of rapamycin on Tgl4 expression. The residual and very moderate positive effect of rapamycin on Tgl4 transcription in Msn4-defective yeast cells argues for the participation of factors other than Msn4 in the transcriptional control of Tgl4 expression by TOR1. Strikingly, orthologs of yeast Msn4p in mammals were reported to constitute a family of early growth response transcription factors, among them the well-known members Egr1 (Krox24), which are known to play important roles in adipocyte differentiation [89, 90] and regulation of cholesterol biosynthetic gene expression [91–93], and Egrs (Krox20) [94]. These data strongly suggest that the regulation of ATGL transcription by the mTORC1-Egr1 pathway is important for the control of lipid metabolism. Otherwise it would not have been conserved along evolution from yeast via Drosophila [86, 87] to mammals [84].

6. Use of Yeast for Drug Target Identification and Validation

Although yeast-based systems are less complicated than mammalian cell-based systems, orthologs of many mammalian proteins are found in unicellular lower eukaryotes. In addition, many other mammalian proteins that do not have sequence homology but do have functional homology can be used to complement functions in yeast. Unlike in higher eukaryotes, the functions of about half of the yeast genes are known on the basis of amino acid sequence similarity with other proteins of known function [95, 96]. This is an enormous resource that is being used for functional analysis. History has shown us that biological mechanisms revealed from the study of lower eukaryotic cells will be applicable to higher eukaryotes. In the future, knowledge of the function of yeast proteins will help in elucidating the function of many mammalian proteins. The similarity between living organisms was noted by Jacques Monod when he said “What is true for Escherichia coli is true for the elephant, except more so” [97]. In the simplest approach, functional complementation can be used to derive a screen in which the activity of the heterologous gene is essential for survival. This approach has been successful even in cases where protein homology is limited, as long as the relevant biological activity is complementary. Yeast-based systems can also be used to define interactions with other proteins. The more difficult approach is to manipulate the heterologously expressed gene to obtain a surrogate phenotype and create “designer yeast”.

6.1. Complementation of Homologous Target Proteins

Many currently used drug targets for the therapy of T2D are GPCRs, such as the glucagon and GLP-1 receptors (see above). Antagonists have been identified by ligand- displacement assays using mammalian cells or their membrane preparations. Agonists have been generally been identified by functional assays. Yeast-based systems have been adapted to identify agonists and antagonists of GPCRs. The mating factor receptor in S. cerevisiae, Ste2, is similar in structure to mammalian GPCRs. Mammalian GPCR can be used to replace Ste2, so that the GPCR can signal through the mating factor signaling pathway when activated by the GPCR agonist [98]. In order to get efficient downstream coupling with the mating factor pathway kinases, the amino-terminal domain of the G_α protein was deleted [99]. The GPCRs expressed in this manner in yeast are useful for screening for agonists. Coupling can also be obtained with the natural yeast G_α protein [100, 101].

The natural ligands of GPCRs can be large peptide ligands, such as for the GLP-1 receptor (see above). In mammalian cells, these GPCRs can be activated by these peptides as well as their derivatives. Antagonists are identified by finding compounds that can displace these peptides. Short peptides can be coexpressed with GPCRs in yeast to develop functional antagonist screens as well as for identifying ligands for orphan GPCRs [102]. Orphan GPCRs are those receptors that have been cloned by sequence homology to known GPCRs but whose function and natural ligands are not known. Peptide libraries have been expressed with secreted sequence tags that are secreted across the plasma membranes of yeast where they come into contact with the GPCRs. Using these peptide libraries, those peptides that specifically interact with and activate the receptor in an autocrine fashion can be identified.

Potassium channels are also important drug targets for the therapy of T2D, among them the ATP-dependent potassium channel (K⁺-ATP) constituted by pore-forming Kir and ligand-binding SUR subunits [103, 104]. Functional screens for K⁺ channel openers and blockers involve expensive equipment and are technically difficult to perform [105]. Therefore simpler assays for developing high-throughput screens have been appreciated. A simple functional screen was developed in S. cerevisiae using complementation of the TRK1/2 potassium transporter knockouts [106]. The inwardly rectifying potassium channel IRK1 has also been expressed in yeast to complement the Trk transporter defect. In the strain expressing the IRK1 channel, the ion channel activity correlates well with the growth phenotype and with patch clamp experiments in Xenopus oocytes expressing these channels.

The influenza M2 channel has been expressed in S. cerevisiae [105]. The influenza M2 channel is a proton channel that is expressed in infected cells. Its function is to increase the acidity of the milieu in which the virus sheds its capsid. When expressed in S. cerevisiae, the M2 proton channel increases the permeability of yeast plasma membranes to ions resulting in loss of yeast cell viability. In order to develop a screen to find influenza M2 protein inhibitors, it was expressed from a galactose-inducible promoter. The screen was designed to find compounds that permit growth and rescue the cells from the permeabilizing effects of M2 protein when the growth medium is supplemented with galactose. Channel screens designed in S. cerevisiae have been useful for high-throughput screening. However, it has to be considered that yeast is slowly growing, and expression of channels in this microbe is difficult and time-consuming.

6.2. Expression of heterologous target proteins

Screens to find ligands for steroid hormone receptors such as retinoid receptors, which constitute an important class of drug targets for T2D and cardiovascular diseases, have been designed in S. cerevisiae [107]. Steroid hormone receptors occur intracellularly and are built up from a ligand-binding domain, a dimerization domain, and a transactivation domain. When ligands induce these receptors to homo- or heterodimerize, their transactivation domain binds the specific response elements and activates specific promoters. The dimerization of the steroid hormone receptor followed by binding and transactivation of specific promoters can be studied in yeast. Homodimerization has been demonstrated using retinoic acid receptors, thyroid hormone receptors, and estrogen receptors [108, 109]. Heterodimerization with the RXR retinoid receptor can also be demonstrated [107]. In this system, the RAR retinoid acid receptors respond to a number of retinoids, but RXR responds only to the RXR-specific 9-cis isomer of retinoic acid [107]. Because all mammalian cells have many representatives of the steroid hormone receptor family expressed naturally, yeast-based systems offer cells with a “null” background activity for studying specific interactions.

Tyrosine-specific protein kinases and phosphatases are important drug targets for the therapy of T2D. Cell-free assays have been popular for this class of targets since the enzymes are easily produced by recombinant means and tyrosine-specific phosphorylation of the natural or an appropriate artifical substrate protein is simple to detect using labeled ATP. An alternative method using Schizosaccharomyces pombe has been published that will find inhibitors that are non-toxic to yeast as well as cell-permeable [110]. The prototypic tyrosine kinase, Src, was expressed under the control of the inducible promoter. Induction of Src results in cell death, and growth rescue can be used for the identification of inhibitors. To adapt the screen for identifying phosphatase inhibitors, this system was modified by co-expressing tyrosine phosphatase on a second plasmid. When the kinase and phosphatase are coexpressed, the cell survives the detrimental effects of kinase expression. Tyrosine phosphatase inhibitors can be identified in this system by looking for compounds that selectively kill the strain co-expressing the kinase and phosphatase [110].

6.3. Detection and Analysis of Protein-Protein Interaction

Proteins carry out their function in most cases by interacting with other proteins. The yeast two-hybrid system, developed by Fields and Song [111], has revolutionized the study of protein-protein interactions. In this system, the transcription factor, GAL4 from S. cerevisiae, is used to set up the assay. GAL4 has two domains, a site-specific DNA-binding domain and an acidic region that is required for transcriptional activation. The DNA-binding and activation domains can be coded by separate genes as long as they are brought together in a heterodimer to reconstitute a functional transcription factor. The system is designed so that when GAL4 binds the GAL4-binding domain on the promoter, LEU2 and/or HIS3 are expressed. Functionally competent chimeric proteins can be made that consist of the DNA-binding domain fused to one protein of an interacting pair and the activation domain fused to the second protein of the interacting pair. Interaction of the proteins that are constructed as chimeras of the activating and DNA-binding domains allow the yeast to grow in the absence of histidine and leucine, thus providing a selective advantage. The yeast two-hybrid system is widely used for identifying homo- and heterodimerizing proteins as well as to develop screens to find compounds that can block two proteins from interacting with each other [112].

The yeast two-hybrid system has been modified to measure the dissociation of interacting proteins by using the URA3 reporter [113]. Yeast cells expressing URA3 can grow in medium without uracil. When 5-fluoroorotic acid (FOA) is introduced into the medium, URA3 expressing cells take up FOA and transform it into a toxic compound. Thus the expression of the reporter gene is toxic and provides a powerful selection procedure. This FOA system is used in the “reverse two-hybrid” system, providing a selective growth advantage and a more powerful system for screening. In the “reverse two-hybrid system”, the interacting protein is expressed inducibly, and only when the interacting proteins are blocked do the cells survive. GAL4 and LexA transcription factors are most often used in the yeast two-hybrid system. In two-hybrid screens, it is useful to have two separate reporter constructs to help in sorting “hits”. Reporters such as Leu2 and LacZ can be expressed in the same cell.

The yeast two-hybrid system has been used to develop screens for ligand-receptor interactions, including peptide hormone receptors, such as the GLP-1 receptor, and the receptor tyrosine kinases, such as the insulin receptor [114–116]. Specific and reversible ligand-receptor interactions between growth hormone and growth hormone receptor, VEGF and KDR, can be studied using the yeast two-hybrid system. Ligand-dependent receptor dimerization can also be studied  using three expression plasmids in which the receptor is expressed as a fusion protein with both the DNA-binding protein as well as the activation domain. The ligand is expressed from a third plasmid. When the ligand binds the two receptors, the DNA-binding domain and activating domains are pulled together and GAL4 is activated.

The yeast two-hbrid system has been adapted to study protein-protein, protein-RNA, protein-DNA, and protein-small molecule interactions [117]. A one-hybrid system has been developed that utilizes cis-acting sequences to identify DNA-binding proteins that can initiate transcription [118]. A yeast three-hybrid was developed to study RNA-protein interactions that are especially useful for developing screens against viruses [119]. In this system, the hybrid RNA containing sites recognized by the RNA-interacting proteins links the two-hybrid proteins containing the DNA-binding and activation domains, respectively. The yeast two-hybrid system has been recently applied to find inhibitors of the N type calcium channel [120, 121]. Alternative screening techniques use mammalian cells to measure calcium channel activity with electrophysiological and spectrophotometric methods to measure calcium influx. These methods are labor intensive, difficult, and not compatible with high-throughput screening. In the yeast two-hybrid system, the interacting, regulatory portion of the α1 subunit of the channel fused to the Gal4 activation domain and the full length β3 subunit fused to the yeast Gal4 DNA-binding domain were expressed. The system could be adapted to find inhibitors of specific calcium channels by selecting the specific interacting domains.

7. Conclusions

Unicellular lower eukaryotes provide an alternate platform for high-throughput screening. Those systems are inexpensive to run and screens can be developed rapidly. Many modular systems are available that are adaptable to important classes of drug targets, such as GPCRs, single-transmembrane receptors such as growth factor tyrosine kinases and phosphatases and ion channels, which are of critical importance for the therapy of metabolic diseases, such as T2D. Functional screens are becoming necessary for developing screens for proteins whose biological functions are not yet known as well as on proteins that interact with new proteins. Because of the ease with which new targets can be explored, functional cell-based screens are becoming the preferred method for finding leads for drug discovery. Test systems based on unicellular lower eukaryotes provide simple and cost-effective means for the identification and validation of drug targets and, most importantly, for the discovery of novel drugs by functional screening.

As thousands of new potential drug targets from genomic information and protein interaction studies have meanwhile been identified, the future of screening is in using chip technology [122, 123]. Systems based on unicellular lower eukaryotes are especially suited for the delivery to microchips. Yeast is robust and is easy to handle and distribute. Thousands of yeast cells can be deposited on chips in a high-density formate. In addition, the surface charge on S. cerevisiae could be used to array the organisms into the desired formate. Currently, the limitation is the sensitivity of reading colorimetric reporters, and alternatives are being investigated. Biosensors and transducers could be used to detect thermal, immunologic, or optical changes [124, 125]. Glucose sensing amperometric systems are being used in clinical microbiology and could be developed for high-throughput screening [126]. Each of the microbial systems described can be adapted to use the reporter that is suitable for the high-density formate on chips for screening. Consequently, a yeast genetic system has been designed for the identification of small-molecule inhibitors of protein-protein interaction on the basis of nanodroplets [127].

Conflict of Interest: The author declares no conflict of interest.

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DOI: 10.31038/EDMJ.2019322

Abstract

Background: Current therapeutics do not provide optimized care in early stage type 2 diabetes and there is no established treatment for prediabetes. We did a randomised trial to assess the efficacy and safety of a popular Ayush product, Sugar Balance® (sugar balance) in patients with newly diagnosed type 2 diabetes and prediabetes.

Methods: In this multicentre, double blind placebo-controlled trial, we randomly assigned (1: 1) eligible participants, 30 years of age or older, of either gender and diagnosed with type 2 diabetes (HbA1c ≥ 6.5%) or prediabetes (FPG ≥ 5.6 mmol/L). We used computer-generated ramdomisation, stratified by type of diagnosis (prediabetes or diabetes). Active recipients were to receive 250 mg capsules containing 3 Organic certified whole herbs: Coccinia indica, Bougainvillea spectabilis and Madagascar periwinkle (sadabahar). The recommended dose was 2 capsules twice daily. The primary outcome was ≥0.5% reduction in HbA_1c for type 2 diabetes subjects and an increased incidence of normalization in impaired fasting blood glucose for prediabetes subjects at 6 months after randomization. The primary analysis included all subjects who received the allocated intervention and had 1 follow up visit after randomisation. All subjects received standard life style recommendations for diet and exercise. Type 2 diabetes subjects were allowed to be on biguanides (metformin) of their choice. This trial is registered with ClinicalTrials.gov, number NCT02866539 and clinical trials registry of India, number CTRI/2016/11/007435, and is completed.

Findings:Between December 14^th 2016 and 28^th August 2017, 122 subjects were randomly assigned to active sugar balance (n=61) or matching placebo (n=61), of whom one subject (assigned to placebo) did not receive the allocated intervention. All follow up was completed on 16^th December 2017. At 6 months type 2 diabetes subjects allocated to active arm had a mean reduction of HbA_1c of -0.52% versus -0.43% among placebo arm (difference of -0.09% 95% CI -0.1705 – -0.0095). 13 (52%) versus 4 (15.4%) prediabetes subjects receiving the polyherbal achieved normalization of impaired fasting glycaemia (<100 mg/dl) (difference of 36.6% 95% CI 10.683 – 56.805). None of the study participants experienced significant hypoglycemia or significant changes in their safety clinical biochemistry parameters on therapy.

Conclusions: Sugar Balance promises to be effective and safe supplement for type 2 diabetes subjects to achieve a better optimization of HbA_1c when taken along with standard dose of metformin and for prediabetes subjects to achieve their target fasting blood sugars with diet and exercise.

Keywords

Ayush, Polyherb, Organic Sugar Balance, Prediabetes, Type 2 Diabetes Mellitus

Introduction

Diabetes mellitus is a non-communicable disease of rising concern in global health. In 2017, more than 425 million people across the globe were diagnosed with diabetes and an additional 350 million with prediabetes [1]. The disease contributes to 10.7% of global deaths [2] and 33 million years lived with disability (YLD) [3]. 77% of these patients live in low- and middle-income countries and have limited access to biomedical health care [1]. India is one of the countries with largest diabetes population [4] and should explore locally available alternate Ayush based treatments to expand access to care.

A recent study suggests that if epidemiological trends continue, achieving the global target of preventing the rise in diabetes patients has a probability of <50% in most of the countries including India [5]. Prevalence of prediabetes is a major threat in India and there are no effective therapies proven for normalization of elevated fasting glucose.

High-quality, low-cost solutions adapted to the local context are critical to addressing the current crisis. Herein we examine the effectiveness of a polyherbal medication, based on the ancient Indian practice of Ayurveda, in the reduction of hyperglycemia in patients with newly diagnosed type 2 diabetes or prediabetes and at two multispecialty hospitals in India.

Holistic medicine is gaining more importance in the treatment of chronic conditions. It is trying to prevent the disease and better the quality of life and health by connecting body, mind and spirit [6]. Ayurveda encourages the practice of emphasizing harmony in the body and cleansing the body of substances that can cause disease [7].

Nevertheless, the most significant reduction in diabetes incidence has been achieved with diet and exercise [8]. Moreover, since Ayurveda-Indian ancient medical traditions- is working on human health by a combination of knowledge, practice and medicine [6].

The present study tested the effectiveness of a polyherbal medicine through a randomized double blinded placebo controlled clinical trial to determine its efficacy and safety in patients with newly diagnosed type 2 diabetes or having laboratory confirmed prediabetes.

Methods

Study Design and Participants

This was a double-blind, two-arm, multicentre placebo controlled randomized trial two sites in India; Mazumdar Shaw Medical Centre Bangalore and KRM Hospital, Lucknow. The trial was designed by Department of Clinical Research at Narayana Health, Bangalore. Ramdomisation support was offered by the Department of Biostatistics at St John’s Medical College and the statistical analysis plan was further verified by data scientists at Boston Children’s Hospital, Boston, MA. The trial participants were randomised under 2 strata based upon their diagnosis; prediabetes or diabetes.

Eligible participants both male and non-pregnant female subjects, 30 years of age or older diagnosed with prediabetes or Type II diabetes by one of four methods: fasting glycaemia >6.9 mmol/L, or a 2 hour post-prandial glycaemia >11.1mmol/L post 2 hour oral glucose tolerance test, or a glycosylated hemoglobin (HbA1c) ≥ 6.5%, or patients with an episode of classic symptoms of hyperglycaemia or hyperglycaemic crisis and a single random glycaemia of >11.1mmol/L in the last 12 months were included .

Eligible participants were excluded if they reported a history of diabetes onset more than 24 months from the date of randomization or reported treatment with any oral hypoglycemic agents (other than Metformin) or insulin, or with any history suggestive of microvascular or macrovascular disease. Additional exclusion criteria included impaired renal function defined as eGFR less than 60 ml/min/1.73m2, blood pressure fluctuations exceeding 10 mmHg on two subsequent clinic visits or known history of hypotension or bradycardia in last 6 months or taking 2 or more antihypertensive medications regularly in the last 6 weeks.

Additionally, women of childbearing age who were unable to use any form of contraception, and any participant participating in another clinical trial with an active intervention or drug or device within the last 60 days, any current or former employees of Organic India Company which manufactures the polyherbal medicine, and any subject refusing consent or unable to commit to treatment adherence were excluded.

Ethical Consideration

This study was approved by the institute ethics committee at Mazumdar Shaw Medical Center (approval number NHH/AEC_RC_2016-078(B) and at KRM Hospital, Lucknow (approval number IEC/02/19/17). The study was conducted in accordance with the International Conference on Harmonization guidelines for Good Clinical Practice (ICH E6); The Code of Federal Regulations on the Protection of Human Subjects (45 CFR Part 46); and The AYUSH Guidelines for Good Clinical Practices. Written informed consent was obtained from all the participants before entry in the study. All potential participants were given a study information sheet and were informed about the protocol procedures, the frequency of follow up and total study duration. All personnel involved in the conduct of this study completed human subject’s protection training.

Randomization and Masking

All eligible subjects were stratified into two groups by block randomization based on the diagnosis of either prediabetes or diabetes. Research personnel randomised participants via a digital randomization phone service at the coordinating center situated at the Department of Clinical Research at Narayana Health, Bangalore, India. Randomization codes were generated by an independent statistician. Packed, blinded medication with a unique number was sent to the recruiting sites. Once randomization was assigned to the treatment or control groups, site personnel received a drug code and issued blinded medications to the patient. The treatment and placebo capsules were identical and packed in identical packaging.

Procedures

The population for the study was derived from subjects referred to KRM Hospital and Research Centre in Lucknow and Mazumdar Shaw Medical Center of Narayana Health Center. Participants were screened for eligibility based on inclusion criteria at executive health checkup clinics, outpatient specialty clinics, and from existing records in the two hospitals. Additionally, caregivers accompanying subjects coming to clinic visit were also invited to participate.

The intervention is a popular polyherbal capsule Sugar Balance® (sugar balance) which is manufactured by Organic India.The active capsule contains a combination of 3 whole herbs: Coccinia indica, Bougainvillea spectabilis and Madagascar periwinkle (sadabahar) of 250 mg having organic certification (ORG/SC/1310/001378) by Control union. The batch number was VX-262 and medication series 358. All three products were as per the organic category of NPOP-Organic Standard of India (National Programme for Organic Production) and NOP-Organic Standard of US (National Organic Programme).

The placebo capsule contained only starch powder of 250 mg, the batch number VX-269 and Medication Series 468. As a standardized approach both the active and placebo products were similar looking Opaque capsules.

Participants were instructed to take two capsules twice a day at a fixed time in the day. Capsules were prescribed to be taken orally after food. There were no diet restrictions, but the subjects had to maintain a diet diary for the entire duration of the study to assist in 24-hour dietary recall. They were stratified as diabetes or prediabetes. All diabetes subjects were allowed to be on standard doses of biganides (metformin) as per clinician choice. All prediabetes subjects were not taking any standard medications for lowering blood sugar but received standard lifestyle recommendations. During the six-month study period, participants had a total of 8 clinical visits during which data were collected: baseline at 0 weeks, follow-up at 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and at 24 weeks.

All participants were given ADA-guided lifestyle advice with free nutrition counselling at each visit. During the study period, the subjects were called every month with reminders about upcoming follow-up clinic visits.

Laboratory procedures were conducted locally in National Accreditation Board for Laboratories of India (NABL) accredited laboratories for study subjects during their scheduled visits, all outcome measures were collated and sent to the data management center.

Study Endpoints

A reading of fasting glycaemia of more than 6.9 mmol/L or an HbA1c value of more than 6.5% is the primary criteria for the diagnosis of type 2 diabetes. Prediabetes is defined as fasting glycemia 5.6 to 6.9 mmol/L. The primary outcome for prediabetes was proportion of subjects achieving a target fasting glycemia of <5.6mmol/L. Safety parameters considered as any significant (2x) change in renal function and hepatic function at any of the follow up visits and at end of the study treatment period.

Statistical Analysis

The main outcome measures, HbA_1c and fasting glycemia were measured at baseline, 90 days (12 weeks) and 180 days (24 weeks) after randomization. Analysis of study data was done using R software version 3.4.4. A secondary analysis was performed after stratification by diabetes status. We used a two-sample t-test and linear regression model to compare numerical variables between the intervention and control groups. A paired t-test was used to compare within-group variables. A Chi-square test was used for categorical variables. All testing was done at a significance level of alpha = 0.05.

Data scientists based in Boston children’s hospital Boston, MA, prepared the analysis plan, and validated the statistical analysis performed by statisticians of Narayana Hrudayalaya.

The analysis was simultaneously done by 2 independents data scientists from India and Boston and the final results were compared.

Data Management Process and Software Used: The data was collected via electronic data source application. Using an Apple Ipad to capture data offline. The application was designed by CliniOps, Inc based upon the data dictionary created by trial scientific expert committee. The software was validated to ensure the proper functioning of application. The application can be used both offline and online and can be accessed on the Web. Data was entered by authorized and trained clinical trial coordinators only.

All the data was collected real time during each visit on to the CliniOps Trial Application version 94.0(electronic CRF) installed on an Apple Ipad 2 offline and synchronized to a cloud server when connected to the internet.

Results

Between December 14^th, 2016 and 28^th August 2017, 200 subjects were screened at two hospitals in India. 122 subjects were randomised, but 1 subject withdrew consent from trial without taking any medications. 121 subjects were included in the intention to treat analysis. All follow up was completed on 16^th December 2017.

The baseline characteristics were comparable between the subjects who received active versus placebo. The average age of the subjects who took active SB was 46.5 (±9.8) years, 93.3% subjects were young <65 years of age. 48.3% (29) were males, 61.6% (37) individuals were either pita-kapha or kapha-vata prakruti as per the ayurvedic phenotype classification.

Out of 60 subjects in the intervention group, 8 lost to follow up. Among them, 3 subjects were from prediabetic and 5 subjects were diabetic. Of the 61 control subjects, 8 were lost to follow up visit. Out of this, 2 subjects were prediabetic and 6 subjects were diabetic. The trial profile of study subjects is represented in figure 1.

Figure 1. Participant flow diagram for Sugar Balance.

Figure 2. Comparison of HbA1c (>6.5%) in diabetic group.

Figure 3. Comparison of Fasting Blood Glucose (>=100 mg/dl) in Prediabetes Group

At 6 months type 2 diabetes subjects allocated to active arm had a mean reduction of HbA_1c of -0.52% versus -0.43% among placebo arm (difference of -0.09% 95% CI -0.1705 – -0.0095). 13 (52%) versus 4 (15.4%) prediabetes subjects receiving the polyherbal achieved normalization of impaired fasting glycaemia (<100 mg/dl) (difference of 36.6% 95% CI 10.683 – 56.805).

Table 1. Characteristics of the study participants at baseline level.

All the participants have Asian ethnicity.

None of the participants have used oral hypoglycemic agents and insulin.

Table 2. Comparison of the mean FBG and HbA1c before and after the intervention among the intervention and the placebo groups.

*p-value was calculated based on Paired t-test

Table 3. Fasting Blood Glucose at Day 180 for Prediabetes

p = 0.008

Safety

All participants were monitored for Adverse Events (AE’s) and Serious Adverse Events (SAE’s) throughout the trial. The distribution of unsolicited AE’s who were administered the active treatment i.e. diabetic participants who were on metformin was 4(14.81%) of 27 and prediabetic was 3(12%) of 25.

The distribution of unsolicited AE’s who were administered the placebo treatment i.e. diabetic participants who were on metformin was 3(11.11%) of 27 and prediabetic was 2(7.69%) of 26. The AE’s reported are Gastrointestinal (GIT) symptoms and dizziness in the diabetic participants and weakness, joint pains and headache in the prediabetic participants in the active and placebo groups. No significant between-group differences were identified (p=1.0).

Using the Diabetes Trial and Safety of Metformin [9], as a reference for safety monitoring, we find that subjects who are diabetic and on the medication of Metformin have a similar AE’s reported i.e. Gastrointestinal (GIT) symptoms (28% in Metformin versus 16% in Placebo), Self-reported GIT problems (9.5% in Metformin compared to 1.1% in Placebo), Anaemia more common in subjects taking metformin (50 subjects) versus 38 subjects in Placebo group.

Discussion

India is home to 75 million people suffering from diabetes and an equally large percentage of those who have prediabetes [10, 11]. Our trial described in this report, used a randomised placebo controlled double blind study to analyze the effects of a polyherbal formulation in bringing about an improvement in glycemic control. Glycated hemoglobin is widely regarded as a standard for monitoring glycemic control in diabetes management. It reflects the mean plasma glucose over period of 120 days, and comprises of the level of blood sugar control in the last 25 days prior to the test. Its fasting value is reflection of the average plasma sugar in the fasting state and also the amplitude of the glycemic spikes occurring post prandial.

The trial participants diagnosed with diabetes were allowed to be on metformin which has an effect of lowering hyperglycemia through reduction of hepatic glucose production and decreased glucose absorption from the intestines. Subjects who received sugar balance along with metformin showed better glycemic control (HbA_1c <0.5%) compared to those only on metformin. The polyherb did not decrease fasting glycaemia when compared to metformin alone. But among the prediabetes cohort, those who were only on sugar balance achieved their target lowering of fasting glycaemia better than the placebo.

Sugar Balance® (SB) contains 3 whole herbs, Coccinia indica, Bougainvillea spectabilis and Madagascar periwinkle or Sadabahar. Coccinia indica has long been traditionally used in Ayurveda for diabetes care and has been studied in vivo extensively for its hypoglycemic effects¹⁰. Phytoconstituents in the plant like polyprenol, steroidalsaponins, and cucurbitane-type triterpenoids are known to intercept glucose metabolism [13]. Bougainvillea spectabilis has also shown antidiabetic properties via increasing glucose-6-phosphate dehydrogenase activity and promoting glucose uptake [14]. Madagascar perinwinkle usage for diabetes comes from the traditional period though its efficacy today has been proven through phytochemical analysis and in vivo research [15]. For example, four alkaloids identified in the plant induce significantly high glucose uptake in pancreatic β-TC6 cells [16].

There is a limited clinical trial information on hypoglycemic effects of individual components of Sugar Balance formulation in Prediabetes and Diabetes patients. Furthermore, proper randomised controlled trial is lacking with all of the polyherbs in combination. Coccina Indica and D-Pinitol isolated from Bougainbellia has been tested for hypoglycemic effect in Humans [12, 17–19]. The Vinca rosea extract is known to have anti-inflammatory properties and hypoglycemic effect in animal models but hypoglycemic action is not tested in clinical trial.

According to a popular textbook published by a Clinical Pharmacologist, Dr. Narendra Singh the potential mechanism of action of Sugar Balance are delayed absorption of glucose, regulation of glucose metabolism as well as improvement in glucose tolerance test [20].

The strengths of this trial is the mimicking of current practice of treating new onset type 2 diabetes with biguanides and prediabetes with just lifestyle and diet. This randomised double-blind placebo-controlled trial, has clinical relevance by proving improvement in glycated hemoglobin among those with type 2 diabetes and increasing the proportion of achieving fasting glycemic control in 6 months. Unlike other trials that have tested the effects of a herbal preparation, this trial tested the intervention along with a standard of care regimen of metformin. The synergistic effect of combining the polyherb with metformin did not increase the risk of hypoglycemia (none reported in the trial). It is important for add-on therapies to help improve glycemic control and not increase the risk of hypoglycemia or cause significant drop in fasting blood sugar. This has been shown to improve long term benefits in diabetes and improves compliance to therapy.

Limitations of this study include the relatively short duration (24 weeks). A longer study might have provided the long-term effect of the combination. However, guidelines limit placebo-controlled trials to 6 months duration. The treatment effect was not significantly different from the placebo group in patients with type 2 diabetes. Probable cause will have been physician preference and patient preference in dose of metformin used through the trial. Also, the lower baseline parameters compared to most other trials could have mitigated the probable greater fall in HbA_1c in a short period. Further studies in this population can be done for seeing efficacy in combination with other oral hypoglycemic agents and also those with moderate to poor control of diabetes.

Conclusion: In conclusion, the result of the present study suggest that Sugar Balance has a potential hypoglycemic action which increases insulin production and decreases the amount of glucose in the blood, and thus could represent a possible preventative treatment strategy for prediabetes as well as new onset type 2 diabetes patients.

Financial Disclosure:Organic India Pvt Ltd are the sponsors of the study, they have funded and contributed to all aspects of the study, and were not involved in any stage of the conduct and analysis of this study, including study design, data collection, data analysis, data interpretation, and writing of the report. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Conflicting Interest: The corresponding author declares that he had full access to the data and complete control on the analysis and publication of its results. He declares having received grants from organic India to carry out this project. He or his family members do not have any financial holding with the company. Other authors except Dr Subramanian Kanan, did receive remuneration from this grant provided by Organic India. Haleh and Leva has no conflict of interest to declare.

References

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DOI: 10.31038/EDMJ.2019321

Abstract

Low Molecular Weight Heparin (LMWH) is commonly used as prophylaxis in the post-op surgical patients but very few adverse effects have been reported. Skin Necrosis is a rare side-effect of Dalteparin therapy and makes up 0.28% [1] of all the reported side effects from its use. This paper focuses on the conservative management of such a case.

Introduction

Dalteparin is commonly used because of its once daily dose for venous thromboembolism prophylaxis with no adjustment required for the weight of the patient. It mainly acts by interacting with the factor Xa and IIa [2]. Skin necrosis has rarely been reported and most cases present within one month¹ of its use and lesions can heal very quickly once the drug has been stopped [3].

The precise mechanism still remains unclear but multiple mechanisms of hypersensitivity have been proposed which include [4]

• Cell mediated hypersensitivity to heparins evidenced by formation of plaques
• Immediate hypersensitivity to heparins appear as palmoplantar pruritis after subcutaneous injections
• Eosinophilia
• Formation of antibodies (Heparin Platelet Factor 4 antibodies) has been linked mostly to the heparin induced thrombocytopenia and cause skin necrosis similar to coumarins.

Skin and provocation tests are contraindicated in patients who have developed skin necrosis or heparin induced thrombocytopenia [4].

Case Report

71 year old patient with a background history of Deep Vein Thrombosis, Pulmonary Embolism and previous Hystrectomy was admitted for a routine Anterior Colporraphy and Posterior Colpo-perineorraphy. As per NICE recommendation she was started on Dalteparin injection for venous thromboembolism prophylaxis 6 hours after surgery. On post-op day three, patient developed painful skin discoloration on both dalteparin injection sites and examination revealed lesions measuring 1.5x2cm and 2×2.5cm on either side of the umbilicus respectively. They were warm to touch with surrounding erythema however patient was apyrexial and systemically well. See Figure 1.

Figure 1a

Figure 1b

Blood work revealed a platelet count of 679/l thus ruling out LMWH induced thrombocytopenia and other relevant blood tests revealed; Haemoglobin 155g/l, White cell count 9.7 × 10⁹/l, International Normalized Ratio 1.0 with completely normal electrolytes. Patient was not taking any antiplatelet or any other anticoagulant at the time. Patient was treated with Loratidine 10mg OD and Ibuprofen 400mg TDS. On post-op day 4, the necrotic area started to develop a blister but the skin on the top remained intact. Patient was advised to keep them covered with sterile gauze pieces so as to prevent the rupture of the blister.

On post-op day 13, patient was reviewed again and examination showed that the lesions were settling down, the one on the right side of the umbilicus had started to reduce in size without any scarring whereas the one on the left was still dark blackish in colour but the surrounding erythema and bruising had settled. Throughout the recovery process patient remained afebrile and only needed paracetamol and ibuprofen for pain control. See Figure 2.

Figure 2a

Figure 2b

Discussion

According to the 2018 NICE guidelines any patient undergoing abdominal surgery should receive low molecular weight heparin for a minimum of 7 days if the venous thromboembolism (VTE) risk outweighs the risk of bleeding³ in conjunction with anti-embolism stockings which produce a pressure of 15-20mmHg at calf until they no longer have reduced mobility [5]. The ACOG and ACCP guidelines state that in case of moderate risk of thromboembolism chemical prophylaxis should be given until the day of discharge [6]. In case of our patient the reduction in mobility was only for the time they remained inpatient for the above mentioned elective procedure and therefore was discharged without a prescription for further LMWH therapy however was advised to keep the anti-embolism stockings on for a period of four weeks. There have been sporadically reported cases of LMWH induced skin necrosis however a life-threatening association of LMWH is Heparin Induced Thrombocytopenia (HIT) which in our case was ruled out by checking the platelet count as soon as the skin lesions were reported on post-op day three and then re-checked on day 8. The following chart was used to assess the risk of HIT (guidance from British Haematology) [7]

The diagnosis of skin necrosis was made on clinical grounds however to confirm the findings, skin biopsy/histology is recommended [4]. In our case we did not do a biopsy nor tested for antibodies as the patient was systemically well and did not develop thrombocytopenia. The option to involve Plastic surgeons in view of possible worsening of necrosis and skin breakdown which might have required skin graft was considered. The safe alternatives to Low Molecular Weight Heparin (such as enoxaparin and dalteparin) are fondaparinux and unfractionated heparin [8] which is an ultra-LMWH and its cross-reactivity to dalteparin is rare [4].

It should be noted that our patient has had deep vein thrombosis and pulmonary embolism following a coronary angiogram in year 2000 for which she received warfarin for one year and had hysterectomy in 2012 for which she did have dalteparin injections for 10 weeks and developed similar lesions and didn’t seek any medical attention at that time as not all the injection sites developed the reaction and these were not as large and painful as the current ones. This suggests that she might have developed sensitivity to LMWH and had an exaggerated response to re-exposure.

Conclusion

Skin necrosis has an association with Heparin Induced Thrombocytopenia and should be investigated as it can be life threatening. Fondaparinux and Unfractionated Heparin can be considered for chemical thromboprophylaxis as safe alternate options in high risk cases. Prompt treatment with Non-steroidal anti-inflammatory agents together with anti-allergics and simple analgesia should be instituted. Early involvement of Dermatologists and Plastic Surgeons should be considered if tissue damage is extensive. In cases of previous skin reactions to LMWH therapy subsequent therapy with a similar agent should be avoided as the spectrum of subsequent reaction may be worse as demonstrated with our case. We do not recommend the use of Antibiotics unless there is clear clinical and biochemical evidence of superadded soft tissue infection.

References

1. Fragmin and Skin necrosis – from FDA reports. April 2018.
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8. Coelho J, Izadi D, Gujral S (2016) Enoxaparin Induced Skin Necrosis. Eplasty 16: 40

DOI: 10.31038/EDMJ.2019315

Abstract

Objective: Several studies have investigated the role of cranberry extract and D-mannose in the prevention of recurrent urinary tract infections (UTIs). The aim of this preliminary study was to test whether the use of a new dietary supplement (UROIAL^TM sachets) containing cranberry extracts (S&R PACs), D-mannose, hyaluronic Acid and Glucosamine Chondroitin may reduce the incidence of episodes of cystitis and improve urinary symptoms.

Methods: In this prospective comparative study, 40 women with an acute diagnosis of cystitis received a single sachet of Fosfomycin Tromethamine (3gr). The subjects were then randomly assigned to two groups: Group A: 20 women were given UROIAL^TM, 2 sachet per day during the first 7 days, then 1 sachet per day for two weeks; Group B: 20 women did not receive any treatment to serve as a control group.

Results: The results of the present study showed a complete remission of urinary symptoms in 37 women, a slight decrease in urinary symptoms was observed in 2 subjects, whereas 1 woman who stopped the treatment was considered a drop-out. Patients in Group A had a lower incidence of episodes of recurrent cystitis during treatment and follow-up; urine samples had significantly lower median bacterial load compared to baseline as well as a symptomatic relief was reported in treated subjects despite the control group.

DISCUSSION: Several studies have investigated and demonstrated the role of cranberry extracts in the prevention of recurrent urinary tract infections (UTIs), on different selected subpopulations and even at increased risk of UTI. Even the use of D-mannose, a natural sugar has proven to have clinical benefit, although clinical data is limited. A randomized placebo-controlled non-blinded clinical trial has shown that a daily dose of 2 g d-mannose was superior to placebo in preventing UTI. Mannose receptors are found on uroepithelial cells lining the urinary tract they constitute the protective mucopolysaccharide layer of the bladder. The mechanism of action involves binding of the mannose receptors to E. coli pili, thus preventing both adhesion to and invasion of urothelial cells. The interaction between mannose molecules and E. coli bacteria can then be washed away with urine voiding.

CONCLUSIONS: Our data indicate that the addition of GAGs (hyaluronic acid and chondroitin sulfate) to cranberry/D-Mannose containing products could represent a valid novel therapeutic approach for the treatment and/or prophylaxis of cystitis.

Keywords

Uncomplicated UTI, Cranberry, D-Mannose and GAGs

Introduction

Urinary Tract Infections (UTIs) are among the most common infectious diseases and almost exclusively caused by bacteria (1). UTIs refers to the presence of a certain number of bacteria in the urine (generally > 10⁵/ml) and symptomatic UTIs are classified in order of severity as upper UTI, with infection in the kidney and lower UTI (cystitis) with bacteria into the bladder (1, 2). Clinically, UTIs classification comprises either uncomplicated or complicated cases, depending on the presence of structural or neurological urinary tract abnormalities (3). According to the latest data, UTIs affect nearly half of all women, and it is estimated that around 11% of women aged over 18 have an UTI each year (4–6).

Based on severity can also be classified as uncomplicated or complicated. This distinction is useful to guide the choice and duration of antimicrobial treatment. In particular, uncomplicated UTIs occur in patients with a structurally and functionally normal urinary tract. In contrast, complicated UTIs are characterized by functional or anatomical abnormalities within the urinary tract (7).

Recurrent UTIs occur more commonly in healthy women who have anatomically and functionally normal urinary tracts (8).

In uncomplicated cases, recurrent UTIs are treatable with a short-course of antibiotics, but the inappropriate use of these antimicrobial agents has been accompanied by the rapid spread of resistant bacterial strains (9). The prevalent pathogenic bacteria that cause UTIs are Gram-negative pathogens and the most frequently isolated bacterial strain is Escherichia coli, representing >80% of infections (10,11).

Bacterial cystitis (also called acute cystitis) can occur in both women and men and some people develop recurrent infections of the urinary tract (12). Three or more urinary tract infections within 12 months define the recurring UTI, as well as two or more recurrences within 6 months. The same bacterial species that caused previous infection is typically responsible for relapses. Approximately 20–30% of adult women with an initial UTI will experience a recurrence within 3–4 months.

The aim of this preliminary study was to verifying whether the use of a new dietary supplement (UROIAL^TM sachets) containing type-A proanthocyanidins (S&R PACs), D-mannose, hyaluronic Acid and Glucosamine Chondroitin can be of use in reducing the incidence of episodes of recurrent cystitis and the elimination of urinary symptoms.

Material and Methods

Study Design

A randomized parallel group intervention trial was designed to evaluate the prophylactic effects of a novel formulation of cranberry extracts combined with d-mannose and hydronic acid on female subjects with a history of recurrent UTIs presenting with uncomplicated cystitis. Of 42 eligible women, two were excluded because they did not meet the inclusion criteria (Fig. 1). Thus, 40 women were randomly allocated and 20 treated with Cranberry, D-Mannose, Vitamin C and Hyaluronic acid; Group A. The duration of the intervention was 12 weeks and the end of follow-up was 3 months after the start of treatment. All participants attended regular clinical appointments.

Figure 1. Flow diagram of enrolment, intervention allocation and data analysis.

Subjects

Eligible subjects were all adult Caucasian females affected by acute uncomplicated cystitis and all had a history of recurrent UTIs. The participants were enrolled (recruitment and treatment) between August 2017 and December 2017 and after antibiotic treatment with Fosfomycin 1 sachet (UROFOS®), were randomly assigned to the two different groups. The inclusion criteria were as follows: (1) two or more episodes of UTI in the last 12 months documented by urine culture; and (2) at least one urinary symptom and/or positive urinary nitrate test or leukocyturia. Women were ineligible if any of the following criteria were present:

(1) pregnancy or lactation; (2) abnormalities of the upper urinary tract, including the presence of urinary stones; (3) a permanent urinary catheter; (4) complete urinary incontinence; (6) stage 5 chronic kidney disease (glomerular filtration rate (GFR) < 15 ml/min). The study took place at the University of Magna Graecia (CZ), Italy.

Nutraceutical Administration

The tested compound is a commercially available as a dietary supplement marketed in Italy, UROIAL (S&R Farmaceutici S.p.A, Bastia Umbra, Perugia, Italy) containing the following ingredients per dose respectively: S&R PACs (250mg) with type-A proanthocyanidins (72 mg), d-mannose (1000 mg), chondroitin sulfate (200mg), vitamin C (120 mg) and hyaluronic acid (100mg). Patients were advised to take 2 sachets for 2 weeks and one sachet for another two weeks.

Laboratory Assessments

Urinalyses and cultures were performed using standard methods, as previously described (13). Diagnosis of UTIs was based on 105 colony-forming units (CFU) in 1 ml of urine sample. Urinary specimens were streaked for isolation on MacConkey agar and blood agar plates for bacterial separation. Positive urine culture results, Gram staining and morphology of pre-dominant colonies were defined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).

Statistical analysis

Clinical data (bacterial count) were analysed by the non-parametric One-Way ABnova Analysis. p value < 0.05 was considered statistically significant.

Results

The study included a total of 40 women with acute cystitis and a history of recurrent cystitis episodes. All but one patient had a complete remission of urinary symptoms after antibiotic treatment with fosfomycin. A slight decrease in urinary symptoms was observed in 2 subjects, whereas 1 woman who stopped the treatment was considered a drop-out. In fact, 38 women participants completed the 12-week intervention period (flow diagram). Table 1 shows the number of symptomatic episodes and number of recurrences recorded during the observation period starting from week 4. Patients in Group A had a lower incidence of episodes of recurrent cystitis during treatment and follow-up and urine samples had a significantly lower median bacterial load compared to baseline. Group A showed a greater number of negative urine cultures compared to group B. Indeed, a higher microbial count (105 and/or >105 CFU/ml) was more frequently observed in the urine cultures of group B (Table 2). Complete symptomatic relief was reported in the majority (85%) of patients in Group A while only 10 % was reported by subjects in the untreated control group.

Table 1. Number patients with symptomatic episodes and recurrences of cystitis during follow-up (12 weeks)

Table 2. Positive E.coil bacterial Counts during follow-up 12 weeks.

Discussion

Several studies have investigated and demonstrated the role of cranberry extracts in the prevention of recurrent Urinary Tract Infections (UTIs), on different selected subpopulations and even at increased risk of UTI (14). Cranberries contain a high content of type A pro-antho-cyanidins whose urinary metabolites interfere with the adhesiveness of uro-pathogenic bacteria to the bladder epithelium, thus preventing adherence of bacteria to the bladder lining (15, 16). They are not as effective as antibiotics and have variable intestinal absorption (17). One RCT showed that both cranberry tablets and juice were able to significantly reduce the proportion of women experiencing rUTI over the 12 months compared with placebo (18% vs. 32%) (18). A Cochrane database systematic review updated in 2012 looked at 4,473 participants (19). It showed that there was no significant reduction in the occurrence of symptomatic UTI overall or for any subgroups tested (20). These included children, the elderly, women, pregnant women, cancer patients, neuropathic bladder patients or spinal patients. The review concluded that with additional studies included the evidence for cranberry juice in preventing UTIs was less than previously thought. There were, however, three studies that showed cranberry products were equivalent to antibiotics in preventing UTIs, with less side effects. These were limited by the lack of standardization between tablets and powders and the limitations associated with lack of information about the active ingredient present in each sample. No recommendations were able to be made.

Even the use of D-mannose, a natural sugar has proven to have clinical benefit, although clinical data is limited. A randomized placebo-controlled non-blinded clinical trial has shown that a daily dose of 2 g d-mannose was superior to placebo in preventing UTI (21). Mannose receptors are found on uroepithelial cells lining the urinary tract they constitute the protective mucopolysaccharide layer of the bladder. The mechanism of action involves binding of the mannose receptors to E. coli pili, thus preventing both adhesion to and invasion of urothelial cells. The interaction between mannose molecules and E. coli bacteria can then be washed away with urine voiding (2, 21, 22).

Vitamin C can acidify urine and has been weakly associated with decreased risk of UTI (23). There is data to suggest that vitamin C can also have a bacteriostatic effect and be effective in treatment and prevention of UTI in pregnant women (24).

Moreover, recently several non-antimicrobial options for rUTIs have been introduced, such as oral or Intra-vesical instillation of glycosaminoglycans (GAGs), which are able to strengthen bladder defense mechanisms. In particular intravesical administration of these GAGs, in particular hyaluronic acid (HA) and chondroitin sulfate (CS), have proven to be successful for both interstitial and recurrent bacterial cystitis (25–27). Glycosaminoglycan (GAG) layer replenishment con be exploited for many indications, including interstitial cystitis, overactive bladder syndrome, radiation cystitis and prevention of rUTI. At present, intra-vesical therapies are reserved for only those with the most unresponsive rUTIs (26). Previous studies by our research group looked at 57 women with rUTI and randomized them to either 50 mL of combination 1.6% hyaluronic acid and 2% chondroitin or treatment with 50 mL placebo. The treatment arm significantly reduced UTI rates over the 12-week follow-up (−86.6% vs. −9.6%) (28). Thus, a management plan including a combination of a non-antimicrobial and selective antimicrobial regime should be considered, such as a novel formulation under investigation in the present study.

Conclusion

Management of UTI should be individualized and symptom-based, but tends to follow a progressive therapeutic ladder. Clinicians should remember to include alternative therapeutics for support and management that should be aimed at improving quality of life (29).

This pilot study reveals that the nutraceutical formulation containing type A PACs from Cranberry extracts, D-Mannose, Vitamin C, Hyaluronic and Chondroitin sulfate (UROIAL®) is a valid therapeutic approach in reducing the incidence of episodes of recurrent cystitis and in the elimination of urinary symptoms. Our data indicate that the addition of GAGs to cranberry/D-Mannose containing products could represent a valid novel therapeutic approach for the treatment and/or prophylaxis of cystitis.

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