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Chapter 9Multifaceted effects of HCQ in diabetes mellitus Introduction Both chloroquine

Chapter 9Multifaceted effects of HCQ in diabetes mellitus
Introduction
Both chloroquine (CQ) and hydroxychloroquine (HCQ) have 4-aminoquinoline nucleus. Presence of a hydroxy group at the end of the side chain in HCQ makes it less toxic and more effective than chloroquine (Figure 1). (reference needed) The ability of HCQ to slow the disease progression in rheumatic arthritis (RA) and other autoimmune diseases led to its inclusion in the class of disease-modifying anti-rheumatic drugs (DMARDs). A renewed interest has been generated in HCQ in the last decade due to research focused on its glucose lowering, lipid lowering, antiplatelet, antithrombotic and cardiovascular (CV) protective effects.2-6
-61595291465Figure 1: Chemical structures of CQ and HCQ
The observation of reduced insulin requirement by CQ, first described in 1984 in a patient with severe insulin resistance, suggested that treatment with CQ or its suitable analogues may be a new approach in the management of diabetes.7 Later on, Smith and colleagues reported that the patients with non-insulin-dependent diabetes mellitus showed a significant improvement in their glucose tolerance, which paralleled the severity of their diabetes.8 HCQ showed improved glycemic control in an observational study of 4,905 RA patients. There was a reduced risk of developing diabetes in patients with HCQ use compared to those who never used HCQ.9 (reference needed) Various actions may be responsible for the multifaceted effects of HCQ.
Anti-inflammatory effect of HCQ
Hydroxychloroquine is thought to improve symptoms of systemic diseases by preventing inflammation. Inflammatory markers are significantly elevated in diabetes and in patients at risk of diabetes.10 Interleukin-6 and C-reactive protein (CRP) are two sensitive physiological markers of sub-clinical inflammation, associated with hyperglycemia, insulin resistance, and overt type 2 diabetes mellitus (T2DM).11 Long term use of HCQ has shown favourable effects in reduction of CRP and other inflammatory markers in systemic lupus erythematosus (SLE) and RA patients.12,13 The mechanisms by which HCQ helps to control pathogenic inflammation are poorly understood but the anti-inflammatory properties of HCQ are attributed to the inhibition of tumor necrosis factor-alpha (TNF-?) and other cytokines and inhibition of leukocyte activation. Figure 2 displays different types of inflammatory markers.14,15
Figure 2: Inflammatory markers that play a role in type 2 diabetes

Various novel mechanisms of action underlying therapeutically relevant anti-inflammatory effects of HCQ are:
Inhibition of the ion channels (Ca++ activated K+ channels)
Ion channels are considered key determinants in the leukocyte biology. Among others, the Ca++ activated K+ channels are believed to promote pathogenic inflammation. Furthermore, NLRP3 inflammasome has been shown to play a key role in promoting atherosclerosis as well as T2DM. The inhibition of Ca++ activated K+ channels by HCQ may lead to impaired inflammasome activation. Also, in vitro studies show that HCQ inhibits ATP-induced caspase 1 activation and secretion of the mature form of interleukin-1 beta (IL-1?) in macrophages. In vivo, this translates to inhibition of caspase 1-dependent neutrophil recruitment by HCQ.16 This novel mechanism has implications in both anti-rheumatic as well as metabolic (anti-diabetic and CV protective) benefits of HCQ.

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Inhibition of endosomal NADPH oxidase (NOX)
NOX enzyme complex is involved in numerous proinflammatory signaling cascades. In particular, signaling of TNF? via TNF-receptor 1 (TNFR1) and IL-1? via IL-1R are mediated in part by uptake of the ligand-receptor complexes into the endosome, activation of endosomal NOX and generation of superoxide and subsequently other reactive oxygen species (ROS). Inhibition of endosomal NOX massively reduces downstream activation of NF?B via these pathways. But, signaling still proceeds with reduced intensity indicating that the endosomal route accounts for part of the cytokine effects.
HCQ has high affinity to acidic compartments, i.e., lysosomes and endosomes. HCQ blocks a signaling pathway common to TNF?, IL-1? and antiphospholipid antibody (aPL), which depends on activation of endosomal NOX2 and leads to proinflammatory and procoagulant cellular responses. Since signaling endosomes serve as physical platforms for crosstalk between different signaling pathways, this might explain the apparently heterogeneous therapeutic profile of HCQ. As a lysosomotropic weak base, HCQ is rapidly protonated, thereby increasing the pH of endolysosomal vesicles. This may block lysosomal enzymes that need an acidic pH. As a consequence, fusion of endosomes and lysosomes is prevented. Inhibition of endosomal NOX2 can explain reduction of cytokine production and plasma concentrations or inhibition of different immune effector cells by HCQ. This effect of HCQ provides an explanation for its beneficial role in the prevention of thromboembolic events.17
Selective inhibition of extracellular oxidants liberated from human neutrophils
Reactive oxygen species produced by neutrophils can exert pro- or anti-inflammatory effects, with respect to their extra- or intra-cellular location. External oxidants may increase the risk of tissue damage, block resolution and lead to permanent inflammation. On the other hand, oxidants inside neutrophils would not be affected, as they are involved in intracellular signaling and can suppress inflammation. The optimal antioxidant should thus preferentially decrease external oxidants. The anti-inflammatory drug, HCQ causes selective inhibition of extracellular oxidants in neutrophils.18
In isolated human neutrophils, treatment with HCQ decreased the mobilisation of intracellular calcium, reduced the levels of external oxidants and diminished the phosphorylation of Ca++-dependent protein kinase C isoforms PKC? and PKC?II, which regulate activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on plasma membrane. On the other hand, no reduction was seen in intracellular oxidants or in the phosphorylation of p40(phox) and PKC?, the two proteins that direct the oxidase assembly to intracellular membranes. HCQ reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug.19
Inhibition of inducible NO synthase (iNOS)
Macrophages produce nitric oxide (NO) via inducible NO synthase (iNOS). Although iNOS was originally isolated from activated macrophages, its expression is induced in many cell types. The NO production by iNOS is responsible for bacterial killing in macrophages. On the other hand, it has also been implicated in many inflammatory diseases with autoimmune background (e.g. vasculitis, lupus, RA). Inhibition of NO production in macrophages may contribute to resolution of inflammation. Perecko et al studied the effect of HCQ on NO production in different macrophage cell types. The results of the study showed that HCQ inhibited NO production in macrophages indicating its anti-inflammatory action in diseases with autoimmune background.20
Glucose lowering effect of HCQHydroxychloroquine has shown favorable metabolic effects on glucose control at both peripheral and pancreatic levels. Clinical and experimental evidences show inhibition of insulin degradation, increase in insulin levels and HbA1c reduction in T2DM patients with suboptimal glucose control.21-23
Inhibition of insulin degradation – At peripheral level
Insulin degradation is a complex process which is not completely elucidated. Insulin is known to have a short plasma half-life of 4–6 minutes due to its rapid uptake and degradation in all insulin sensitive tissues of the body. More than 50% of insulin is cleared in a single pass through the liver.
The initial step in insulin degradation is binding of insulin to the cell membrane mediated by specific insulin receptors. After binding to the receptor, internalization of insulin into endosomes takes place. Once the insulin-receptor complex has been internalized, insulin undergoes rapid degradation through insulin degrading enzymes (IDEs): glutathione insulin transhydrogenase, lysosomal protease, and insulin protease (insulinase). Some insulin is also degraded on the cell membrane in the absence of internalization, and is metabolized by membrane bound insulin protease. It accounts for more than 95% of all insulin degrading activity in human muscle and fibroblast cells (Figure 3).

Figure 3: Inhibitory action of HCQ on insulin degradation

HCQ, is an acidotrophic drug. It selectively concentrates in endosomes causing an increase in pH. Increase in pH inhibits the action of IDEs, and thus insulin degradation. This unique action of HCQ increases blood insulin levels leading to favorable metabolic effects.
26670259715This mechanism of HCQ has been elucidated in an experimental study where HCQ significantly reduced percentage insulin degradation. It was also observed that insulin-deficient experimental models had decreased insulin degrading activity which may be due to a reduction in enzyme synthesis. This may be interpreted as a protective mechanism, such that in the presence of low levels of insulin, less is degraded. HCQ also increased insulin binding to its receptor and altered hepatic insulin metabolism, thereby potentiating insulin action.24
Emami J et al25 have also shown glucose and insulin homeostasis with HCQ in their experimental study. A significant linear relationship between the glucose reduction and HCQ concentration (p<0.001) and HCQ dose (p<0.002) was observed (Figure 4) (both figures depict concentration, figure for dose is needed). HCQ appears to sustain higher insulin levels and hence has therapeutic potential in the treatment of patients who have residual ?-cell function.
Figure 4:
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Improvement in insulin sensitivity – At pancreas level
Insulin resistance comprises one principal aspect of metabolic syndrome, an amalgamation of risk factors that predict CV events. In a pilot intervention study, Mercer and coworkers26 showed that six weeks of HCQ treatment improves insulin sensitivity in obese non-diabetic individuals without a known systemic inflammatory condition. Matsuda Insulin Sensitivity Index (ISI), HOMA-IR were assessed at 0, 6 weeks and 12 weeks (i.e. 6 weeks post stopping HCQ). There was a statistically significant increase in ISI after 6 weeks of HCQ 6.5 mg/kg and a decrease in ISI toward baseline after stopping HCQ (Figure 5).
Figure 5: Effect of HCQ on insulin sensitivity index
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HOMA-IR, a measure of insulin resistance, decreased significantly from 2.1 to 1.8 and crawled back towards baseline after stopping therapy (Figure 6). This degree of improvement in insulin sensitivity may translate into a reduced risk of diabetes.
Figure 6: Effect of HCQ on insulin resistance
2667042545
Further to examine how HCQ affects glucose homeostasis, researchers from the university of Pittsburg (Diabetologia. 2015 Oct;58(10):2336-43) conducted a 13-week trial in non-diabetic adults who had risk factors for insulin resistance such as fasting blood glucose (FBG): 100–125 mg/dL, fasting insulin >7 ?U/ml, history of gestational diabetes or a parent with T2DM. The study findings demonstrated that HCQ improves both insulin sensitivity and beta-cell function (Table 1).

Table 1: Effect of HCQ on insulin sensitivity and beta-cell function
Variable Placebo HCQ P value
Insulin sensitivity SI (10-4 pmol-1 1 min-1) Baseline 0.58±0.42
0.52±0.27 0.60
Change from baseline ?0.11±0.04* 0.08±0.03*
0.001
% change from baseline ?18.4±7.9*
20.0±7.1*
0.001
Beta-cell function Disposition index Baseline 1,214±691
1,721±2,014
0.36
Change from baseline ?218±158*
352±143†
0.013
% change from baseline ?218±158*
45.4±12.3†
0.002
*p<0.05 within group (baseline vs 13 weeks); †p=0.05 within group (baseline vs 13 weeks); SI: Insulin sensitivity
A potential mechanism by which HCQ may influence insulin sensitivity could be modulation of adipose tissue inflammation and adiponectin production. It is known that altered adipokine profile contributes to the development of impaired glucose homeostasis, low-grade inflammation and obesity-related comorbidities.27 Significant increase in plasma adiponectin level after HCQ (400 mg/day) treatment (18.7%) but not after placebo (0.7%) in a study suggests the possibility of anti-inflammatory effects of HCQ in adipose tissue (Figure 7). By increasing adiponectin levels HCQ may enhance the positive metabolic effects not only at the peripheral sites but also at the pancreas. (reference needed)
Figure 7: Effect of HCQ on adiponectin level
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Beta-cell preservation – At pancreas level
Beta-cell apoptosis and ?-cell proliferation are key players in the dysfunctional remodeling of the islets of Langerhans (IOL) and consequent hyperglycemia in T2DM. Early changes in T2DM include IOL hypertrophy/hyperplasia followed by degenerative changes, and their atrophy and infiltration by inflammatory cells, particularly macrophages. Histological and immunohistological results of the study showed preservation of beta cells with HCQ.28
In immunohistological results, most of the cells of IOL of the control group showed an intense widely distributed insulin immunoexpression all over the islets. On the other hand, the IOL of the diabetes mellitus group exhibited fewer less intense insulin expressing cells. Minimal affection of the IOL was denoted in the HCQ + diabetes mellitus group with preservation of most of their insulin expressing cells which appeared numerous and had intense reaction compared with the diabetes mellitus one (Figure 8). Thus, HCQ shows a favorable effect on the structure of endocrine pancreas in a type 2 diabetic model.

Figure 8: Histological and immunohistological findings

299085010160Immunohistological results
a- Control group, b – Diabetes group, c – Diabetes + HCQ group
00Immunohistological results
a- Control group, b – Diabetes group, c – Diabetes + HCQ group
1143003810Histological results
a- Typical view of IOL, b – Disrupted IOL in diabetes group, c – Preservation in IOL in HCQ group
00Histological results
a- Typical view of IOL, b – Disrupted IOL in diabetes group, c – Preservation in IOL in HCQ group

Cardiovascular protective effects of HCQCardiovascular disease due to atherosclerosis is the leading cause of death in chronic inflammatory disorders. This may be due to the adverse effects of chronic inflammation on the vasculature. In an inception cohort of RA patients, treatment with HCQ was independently associated with a 72% reduction in all incident CV disease events and a 70% reduction in the risk of incident composite coronary artery disease, stroke, and transient ischemic attack.29 The biological plausibility of this protective association is supported by the favorable associations of HCQ with beneficial changes in lipid profiles, reduced risk of thrombotic events and platelet inhibitory effect.

Lipid lowering effect of HCQ
Lipid lowering effect of 4-Aminoquinolines is known for long when in 1986, Beynen ll showed that low-dose chloroquine decreased cholesterol synthesis.30 The induction of inflammation provides a link between hyperlipidemia and atherogenesis. HCQ is known to improve inflammatory markers. Several studies have demonstrated the lipid lowering activity of HCQ.31-38
The latest in this series of studies was a study conducted by Pareek A et al.39 The researchers evaluated efficacy and safety profiles of atorvastatin and HCQ combination in comparison with atorvastatin monotherapy in the treatment of dyslipidemia. Eligible patients were randomly allocated to receive either atorvastatin 10 mg or atorvastatin 10 mg and HCQ 200 mg for 24 weeks. Patients were divided into two cohorts: cohort 1 – patients with normoglycemia (HbA1c: 5.7% and taking no anti-diabetic medicine) and cohort 2 – patients with pre-diabetes (HbA1c: 5.7% to 6.4% and taking no anti-diabetic medicine), patients with T2DM (HbA1c: 6.5% or patients taking anti-diabetic medicine). There were significantly greater percentage reductions in low density lipoprotein-cholesterol (LDL-C), non-high-density lipoprotein-cholesterol (HDL-C) and total cholesterol (TC) in patients treated with combination therapy than atorvastatin alone (Table 2).
Table 2: Effect of two different therapies on lipid profile
Lipid profile Mean % change at week 24
ATV ATV + HCQ P value
TC (mmol/L) -24.41 -29.30 0.013
LDL-C (mmol/L) -32.52 -39.54 0.008
HDL-C (mmol/L) +2.20 +7.55 0.129
Non-HDL-C (mmol/L) -30.37 -36.76 0.009
TG (mmol/L) -10.52 -12.72 0.668
ATV: atorvastatin, (+): increase in levels, (-): decrease in levels.

The addition of HCQ resulted in a 5.16% incremental fall at week 12 and 7.02% incremental fall at week 24 in LDL-C along with significant decrease in HbA1c and FBG levels. Significantly more patients achieved LDL-C and TC goals and lesser patients developed diabetes with combination therapy.
Anti-thrombotic effect of HCQ
An apparent effect of HCQ to reduce thromboembolic events has been recognized for more than two decades. Carter et al in 1971, found that HCQ, though not anticoagulant, is an effective agent in reducing deep venous thrombosis (DVT) in the leg after major surgery.40 The incidence of DVT was reduced to 5% compared with an incidence of 16% in a similar untreated group of patients. Further, HCQ was used as a prophylactic that prevented thrombotic events in the postoperative period following total hip arthroplasty.41 Subsequent analyses of clinical data supports the use of HCQ to prevent emboli in thousands of patients following orthopedic procedures.4 Several studies—both prospective and retrospective in SLE have found reduction in thrombosis risk with HCQ usage (Table 3).4
Table 3: Evidences showing that HCQ prevents thrombosis in patients with SLE
Study Study design Thrombosis studies Outcome
Wallace (1987) Retrospective (N = 92) Arterial + venous P < 0.05
Petri et al. (1994) Prospective cohort
( N = 393) Arterial Odds ratio (OR), 0.36
Ruiz-Irastorza et al. (2006) Prospective cohort
(N = 232) Arterial + venous Hazard ratio (HR), 0.28
Tektonidou et al (2009) Case-control (cases = 144, controls = 144) Arterial + venous HR, 0.99
Jung et al. (2010) Nested case-control (cases = 54, controls = 108) Arterial + venous OR, 0.32
Several mechanisms have been proposed for the anti-thrombotic effects of HCQ. Many years ago, HCQ was shown to reduce platelet aggregation in vitro, but this effect was not measured in patients treated with HCQ.4 Platelet aggregation may also be reduced by HCQ through inhibition of the alpha-granule release reaction, but this was limited to in vitro measurements.4
Anti-platelet effect of HCQ
Hydroxychloroquine’s Efficacy as an Antiplatelet Agent (HEAT trial)4 evaluated its anti-platelet effect and compared it head-on with the commonly prescribed anti-platelet therapies in humans. A study showed 11% reduction in platelet aggregation with HCQ and 31.2% reduction on combining it with aspirin. There was also a significant decrease in fibrinogen and erythrocyte sedimentation rate values (Figure 9).
Figure 9: Effect of HCQ on platelet aggregation

Anti-platelet action may be downstream to the production of thromboxane A2 in the arachidonic acid pathway. HCQ’s accumulation in dense granule in platelets may inhibit aggregation by decreasing the secretion of aggregation amplifying substances from platelet granules.
With possible additional beneficial effects over the traditional risk factors of CV disease like hyperglycemia and hyperlipidemia, as shown in other studies, future studies might focus upon the potential of HCQ as an antiplatelet and anti-inflammatory agent for the treatment of CVD.

Conclusion
HCQ is known to exert its anti-inflammatory effect via several mechanisms including inhibition of the ion channels, inhibition of endosomal NOX, selective inhibition of extracellular oxidants liberated from human neutrophils, and inhibition of iNOS. Apart from its glucose-lowering property, it also displays other beneficial actions such as cardioprotection. These factors render HCQ a potentially valuable agent in the treatment of T2DM.

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