DM199 for Pregnancy Complications

Preeclampsia and fetal growth restriction are leading causes of maternal and fetal morbidity. Both result from poor placental function. Preeclampsia is further characterized by inflammation and oxidative stress, leading to maternal endothelial dysfunction and hypertension. Hence, a drug that improves maternal vascular function including vasodilatation (and blood pressure reduction) may be a treatment for both conditions. Tissue kallikrein(KLK1) is an endogenous enzyme that cleaves kininogen to produce active kinins, mainly bradykinin. Bradykinin is a potent natural vasodilator with pro-angiogenic and possible anti-oxidant and potentially anti-inflammatory effects. Specifically, bradykinin binds and activates the bradykinin 2 receptor located on blood vessel endothelium. Activation of these receptors increases nitric oxide and prostacyclin production, resulting in relaxation of the smooth muscle of blood vessels and consequently vasodilation. Bradykinin 2 receptor activation may also have other beneficial actions such as upregulating antioxidant defenses. DM199 is a pharmaceutical formulation comprised of recombinant tissue kallikrein. It is a protein that is identical to KLK1,except for two amnio acids. Given the beneficial actions of KLK1, DM199 could be a therapeutic to treat preeclampsia. Furthermore, its vasodilatory properties might enhance blood perfusion to the placenta. If so, it could have merit in treating fetal growth restriction. Preclinical studies, animal toxicology studies and clinical trials (non-pregnant population) have shown DM199 to be safe and well tolerated. DM199 is a protein meaning it is unlikely to cross the placenta and reach the fetus. These properties make DM199 an ideal candidate to evaluate as a possible treatment of preeclampsia and possibly fetal growth restriction. This protocol describes an unblinded study to determine an effective and safe dose of DM199 for women with preeclampsia and/or fetal growth restriction.Preeclampsia is an unwelcome complication affecting 5-7% of all pregnancies.2 It is one of the two leading causes of maternal death during pregnancy. For every maternal death related to preeclampsia, another 50 to 100 women suffer severe health injuries.3 Around the world, there are estimated to be around 1.6 million cases of preeclampsia with severe features every year.

Preeclampsia is a pregnancy specific disorder that presents with hypertension and multi-organ injury in the second half of pregnancy. A hallmark of preeclampsia is severe maternal vascular dysfunction, where damage to the mother's blood vessels leads to hypertension and injury to many vital organs. The mother is at risk of developing seizures (eclampsia), cerebral injury like infarctions or intracranial haemorrhage, renal injury or failure, hepatic rupture and pulmonary edema. She may also develop haematological complications which include haemolysis, elevated liver enzymes and low platelet (HELLP) syndrome and disseminated intravascular coagulation. This puts her at a high risk of haemorrhage. Preeclampsia can also cause a placental abruption, where the placenta prematurely separates from the uterine wall due to bleeding resulting in catastrophic consequences for both the mother and unborn child.

Preeclampsia is both a placental and maternal disease. In early pregnancy the placenta fails to properly implant in the inner lining of the uterus. This may result in co-existing fetal growth restriction where the fetus fails to reach its genetically pre-determined growth potential. Fetuses with fetal growth restriction are at increased risk of adverse perinatal outcomes including stillbirth.

Preeclampsia is common Preeclampsia complicates about 5% of all pregnancies and is estimated to cause at least 42,000 maternal deaths every year.4,5 Worldwide, it is estimated that over 90% of deaths caused by preeclampsia occur in low and middle-income countries. Globally, preeclampsia disproportionately affects minority populations and those living in low and low middle-income countries. In South Africa, hypertensive disorders of pregnancy are responsible for 14% of all maternal deaths.8 Preeclampsia has no treatment apart from delivery There are certain medications given to women with preeclampsia: drugs to reduce hypertension or an infusion of magnesium sulphate to reduce the risk of eclampsia. However, these medications only control the late end-organ consequences of preeclampsia. Critically, there are no drugs that slow the underlying disease progression (such as improve placental health or quench the blood vessel injury).

Because there are no drugs to treat preeclampsia, delivery is the only definitive treatment. Birth of the fetus arrests the disease because the placenta is removed - the source of the anti-angiogenic factors responsible for the vascular and end-organ damage. Even after delivery, it may take weeks for a woman to recover from the acute end-organ complications of preeclampsia.

Preeclampsia has lifelong implications Having had preeclampsia leaves a lifelong legacy of an increased risk of chronic illnesses for the mother, especially cardiovascular health risk. Over the remainder of her life she has an increased risk of developing chronic hypertension, a 2-4 fold increased risk of stroke, heart and renal failure, and death from cardiovascular disease.2,9 Frequently the baby is born growth restricted, premature, or both: these synergise to incur lifelong adverse health effects for the newborn.2 1.2 Pathogenesis of Preeclampsia Preeclampsia arises in two stages: the first is placental disease which is followed by maternal vascular injury.

Stage 1: Placental disease In normal early pregnancy, the placenta actively remodels the maternal vasculature inside the uterus. Columns of placental cells enter spiral arterioles in the uterus (maternal vessels) and strip them of their muscular walls, rendering them unable to contract. The result of spiral arteriole remodelling is a low-pressure, high-capacity system which is ideal for the transfer of nutrients and oxygen to the growing fetus.10-13 In preterm preeclampsia and fetal growth restriction, uterine vascular remodelling goes awry.14,15 The maternal spiral arterioles retain some ability to contract, resulting in a high pressure, turbulent blood flow..15-17 This injures the placental cells and causes cellular stress of the surface cellular layer of the placenta, the syncytiotrophoblast.

As pregnancy continues into the second trimester, the diseased preeclamptic placenta secretes elevated amounts of anti-angiogenic factors into the maternal circulation that cause the maternal vascular injury seen in stage two of preeclampsia. There are many candidate factors secreted in excess by the preeclamptic placenta that may contribute to blood vessel dysfunction.18 A likely central driver is an anti-angiogenic protein called soluble fms-like tyrosine kinase-1, or sFlt-1.12,18,19 sFlt-1 binds to the pro-angiogenic factor VEGF-1 (vascular endothelial growth factor-1) and renders it inactive. VEGF signalling on blood vessels is necessary to maintain a pro-angiogenic state. Neutralized by sFlt1, VEGF-1 can no longer bind to its specific receptors studded along blood vessels to maintain healthy blood vessel homeostasis.19 In addition to sFlt-1 are many other factors secreted in excess by the diseased placenta into the maternal circulation to exacerbate the maternal vascular damage. These include pro-inflammatory cytokines, exosomes, and other anti-angiogenic molecules.10,20-22 Stage 2: Endothelial dysfunction and maternal vascular disease In the second stage of the pathogenesis of preeclampsia, the placenta releases these anti-angiogenic factors into the maternal circulation.19 This results in vasoconstriction leading to hypertension and damage to maternal end-organs.

There are two main layers of arterial blood vessels. The inner lining is a single layer of endothelial cells that are linked together to form a watertight junction. Below the endothelial layer are vascular smooth muscle cells made up of multiple layers.

The role of the vascular smooth muscle layer is to contract (narrowing the diameter of the blood vessel) and increase the blood pressure or relax (widens the diameter of the blood vessel), reducing blood pressures allowing a greater volume of blood to flow through. Whether vascular smooth muscle cells contract is dictated by signals received from the adjacent endothelial cells.

Endothelial cells are the master regulators of blood vessels and take the lead in coordinating blood pressure and general vascular health. They receive messages from proteins and molecules in the bloodstream and act on them. They can send messages into the bloodstream to be relayed to distant blood vessels. They may also relay local information in the other direction, to the vascular smooth muscle cells lying just beneath.

Specifically, endothelial cells:

1. Receive signals from molecules present in the circulation. These molecules will usually lock onto its specific receptor on the cell surface of endothelial cells. The receptors will relay signals inside the cell to then issue further commands.

   Example of signals include vascular endothelial Growth Factor (VEGF) or placental growth factor (PIGF) (both promote vessel relaxation), or bradykinin (promotes vascular relaxation and a fall in blood pressure) (See figure 2, Endothelial cell signalling). Angiotensin II binds to the angiotensin receptor to promote vascular constriction (increasing blood pressure).

2. Send signals locally to the underlying vascular smooth muscle: Endothelial cells can release molecules that instruct vascular smooth muscles to either dilate or constrict. Three important molecules released by the endothelium are nitric oxide (NO), prostacyclin and endothelium derived hyperpolarizing factor (see figure labelled Endothelial cell signalling). They are the main players promoting local vascular relaxation and reducing blood pressure. Endothelin-1 is another vasoactive molecule, a peptide that is one of the most potent, naturally occurring vasoconstrictors (causing hypertension). Some of these molecules, such as endothelin-1 and nitric oxide may also be released into the bloodstream where they presumably travel to more distant blood vessels to exert their effects.

3. Send signals into the bloodstream: Endothelial cells can release specific molecules into the blood that travel distantly to signal and target other endothelial cells. Examples are sFlt1 and cytokines which cause a pro-inflammatory state leading to endothelial dysfunction.

4. Send signals into the bloodstream: Endothelial cells can release specific molecules into the blood that travel distantly to signal and target other endothelial cells. Examples are sFlt1 and cytokines which cause a pro-inflammatory state leading to endothelial dysfunction.

In preeclampsia, the placental factors released into the maternal circulation (stage 1) cause endothelial dysfunction (stage 2). They switch on molecular cascades causing endothelial cells to issue signals for blood vessels to contract. This leads to hypertension. In addition, vasoconstriction reduces blood flow to many vital organs.

The elevated levels of sFlt1 from the placenta reduces VEGF signalling. The preeclamptic placenta also secretes less PIGF (VEGF and PIGF signalling is desirable as they cause vasodilation).18 The endothelial dysfunction also reduces production and release of nitric oxide, and prostacyclin into the vascular smooth muscle, resulting in vasoconstriction.

The endothelial dysfunction is driven by a pro-inflammatory state (where cytokine molecules signal to the endothelial cells, contributing to the dysfunction) and oxidative stress in the cells. These processes cause the endothelial cells to become dysfunctional, and this contributes to the release of signalling molecules to the vascular system resulting in a pro-hypertensive state.

Thus, endothelial dysfunction causes widespread hypertension and vessel injury. This reduces blood supply to many of the mothers' vital organs and increased passage of fluid out of blood vessels. The net result is hypertension, edema, maternal vascular disease and maternal organ injury.

The severity of preeclampsia varies greatly. It can be mild, where laboratory tests reveal maternal organs to be mildly injured and are expected to promptly recover after the placenta is removed. However, preeclampsia can be very dangerous - where the organ injury is so severe it threatens the life of the mother and unborn baby.

1.3 Treatment of Preeclampsia A drug that can slow disease progression for preeclampsia would be a major breakthrough. It could save many lives and reduce healthcare costs. Unfortunately, no such drug exists.

Without a specific treatment, the approach to clinical management when preeclampsia is diagnosed is to 'watch and wait' and to 'time birth'. We closely monitor the degree of maternal organ damage and if severe injury is apparent, we deliver the baby.

A drug that could quench the underlying pathology of preeclampsia could ameliorate the severe injury to maternal organs. Such a drug could prove useful for all types of preeclampsia:

1. Early preterm preeclampsia: It would be particularly useful in cases of preterm preeclampsia: if the drug can reduce the disease impact, the pregnancy could be allowed to safely continue. Instead of immediately delivering the baby preterm, it could be safely left in the uterus to further develop and reach a more mature gestational age. This could reduce immediate health care costs in the way of avoiding neonatal intensive care admission and present the baby with better prospects of lifelong health.

2. Preeclampsia at late preterm/ term gestation: For preeclampsia occurring at late preterm gestation (diagnosed when the pregnancy has reached at least 34 weeks of pregnancy) we would usually proceed with planning birth soon. Even for these cases, a safe drug that tackles the underlying disease pathology would be useful. This is because by the time preeclampsia is diagnosed, there is often already evidence of severe organ injury23 (preeclampsia with severe features, using criteria published by the American College of Obstetricians and Gynecologists).24 Hence, the drug could quell the disease long enough to buy time - around 24-72 hours - for clinicians to safely birth the baby. It could offer safer outcomes for the mother.

3. Postpartum preeclampsia: The damage caused by preeclampsia takes time to resolve. After birth, mothers may still suffer severe complications of preeclampsia. These complications include very high blood pressures, eclampsia, pulmonary edema and heart failure. Current treatments can improve the symptoms, but better treatments are needed that ameliorate preeclampsia.

4. Fetal growth restriction: Preeclampsia is often accompanied by fetal growth restriction which is a result of uterine artery vasoconstriction and placental dysfunction. If a treatment could reverse the vasoconstriction, it may improve fetal growth and development.

Clinical trials investigating treatments for preeclampsia There have been very few randomised clinical trials of potential therapies to treat preeclampsia. Nearly all have reported negative findings, suggesting the investigational drug was not effective.

The general approach to randomised trials testing investigational agents to treat preeclampsia is to administer the drug to women with preterm preeclampsia. The primary outcome for most trials has been to assess how long preeclamptic women were able to remain pregnant before birth was required, because maternal organ injury became too severe (or the fetal condition deteriorated). Hence, a longer period between randomisation and birth would suggest the drug was able to quench the disease process and allow the pregnancy to safely progress for longer (and for the baby to stay longer in the uterus to develop and mature). At preterm gestations (such as 24-32 weeks gestation), even gains of around 5-7 days are likely to translate into significant health improvements for the newborn and reduce the risk of death, or permanent chronic illnesses.

A number of drugs have been investigated. Sildenafil: A trial of oral sildenafil (50mg, three times a day) given to women with preterm preeclampsia significantly lengthened the pregnancy by 4 days. Unfortunately, since then, a large trial evaluating sildenafil to treat fetal growth restriction was abruptly stopped. It raised concerns that sildenafil was crossing the placenta into the fetal circulation, causing fetal lung injury (neonatal pulmonary hypertension) and neonatal loss.25,26 Hence, sildenafil cannot be considered further.

Metformin: We published a randomized trial in British Medical Journal showing oral metformin may prolong gestation by a week in women admitted for expectant management.27 Metformin is a drug used to treat diabetes, including diabetes in pregnancy. We embarked on this trial in light of our prior laboratory experiments which demonstrated metformin may have actions that improve endothelial function and dilate blood vessels.17 We are now undertaking randomized validation trials at Tygerberg Hospital, Cape Town and a multicentre randomized validation study in Sweden.

Other agents that did not work in randomised trials: Randomised trials of investigational agents that have reported a negative outcome include intravenous antithrombin III (meant to dampen the immune system but a US multi-centre trial failed to find benefit,28 an unpublished Japanese trial also did not find benefit [drug name KW-3357])29; pravastatin30 (a cholesterol lowering drug postulated to have actions that counter preeclampsia31,32), and a randomised trial we did that tested 40 mg of oral esomeprazole33 (drug used to treat gastric reflux, also postulated to have molecular actions that could counter preeclampsia34).

Comanche Biopharma is a US company that is examining a molecular therapy (comanchebiopharma.com). Based on preclinical studies,35 they are testing whether administration of a drug made of short interfering RNA) - (siRNAs) - can enter the placenta and reduce the production and release of sFlt1, one of the main drivers causing the endothelial dysfunction. This concept is early stage: to date, they have not published results of early phase trials.

1.4 Potential of DM199 to treat preeclampsia The ideal drug to treat preeclampsia may be one that 1) is highly efficacious at reducing blood pressure, 2) increases blood flow to the placenta to reduce placental disease, 3) reduces endothelial injury, 4) does not cross through into the placenta and 5) has already been shown to be safe when administered to humans in a non-pregnant population.

DM 199: a promising candidate treatment for preeclampsia DM 199 is a manufactured version of Kallikrein-1 (or KLK1) but with small modifications that increases its stability. It has potent blood pressure lowering effects and does this by switching on existing natural molecular machinery within cells. It can be given as an intravenous dose (short acting), or as a subcutaneous injection (longer acting).

DM 199 is protein drug version of a natural enzyme Kallikrein-1: a key player in blood pressure reduction.

Kallikrein-1 is continually made and secreted from endothelial cells, but also likely released from other tissues, such as the kidneys, pancreas and lungs. DM199 is a recombinant (synthetic) form of Kallikrein-1. The main role of kallikrein-1 is to cleave inactive kininogens to make active bradykinin or lys-bradykinin. Bradykinin or Lys-bradykinin are cell signalling molecules that powerfully reduce blood pressure (See figure 3, Kallikrein 1 and DM 199 signalling).

Lys-bradykinin is a short 10 amino acid peptide that binds to and activates bradykinin 2 receptors that are studded on the surface of endothelial cells. Once activated, bradykinin 2 receptor triggers molecular circuitry within the endothelial cells that upregulates the production and release of two potent molecules that relax blood vessels and reduce blood pressure: nitric oxide and prostacyclin.

Nitric oxide and prostacyclin diffuse into the underlying vascular smooth muscle and causes them to relax. This reduced vascular contraction dilates the blood vessel.36 Nitric oxide and prostacyclin are co-released from endothelial cells and believed to work in synergy. In addition, there is evidence that activation of the bradykinin 2 receptor also promotes release of a third vasodilating molecule, endothelium derived hyperpolarizing factor.37 This also signals to the underlying smooth muscle and causes it to relax, further dilating blood vessels.

DM 199 may mitigate the placental disease - stage one of preeclampsia It is plausible that DM 199 may not only act to protect the maternal blood vessels but could also reduce placental dysfunction - the origin of the anti-angiogenic factors causing widespread vascular damage.

The uterine arteries are the two main blood vessels supplying maternal blood to the uterus, providing oxygen and nutrients to the placenta and baby. The uterine arteries narrow when there is preeclampsia (this narrowing is readily detected using ultrasound where resistance to blood flow increases, measured on Doppler ultrasound as an increase in the 'pulsatility index'). This likely occurs because of the inadequate uterine vascular (spiral arteriole) remodelling during early pregnancy (discussed above) - increasing blood flow resistance.

Nitric oxide in the uterine arteries that results from DM 199 administration could result in uterine artery dilation. Hence, if DM199 can dilate uterine arteries (and also open constricted vessels immediately downstream such as the arcuate arteries and the spiral arterioles in the uterus), it could increase oxygenation to the placenta and fetus, reducing the placental dysfunction. This might reduce the placental injury and reduce the release of anti-angiogenic factors from the placenta, opening a second front to combat preeclampsia and fetal growth restriction.

DM 199 may have potential to treat fetal growth restriction If placental rescue happens via dilation of the uterine arteries, there may be direct fetal benefits. Placental disease and low oxygenation restrict fetal growth (causing fetal growth restriction). Indeed, due to their shared origins as placental diseases, fetal growth restriction and preeclampsia often co-exist.

If we show in our planned preeclampsia trials that using DM 199 promotes uterine artery dilatation on ultrasound, it may mean placental and fetal rescue may be occurring. If so, DM 199 may have merit as a treatment for fetal growth restriction, in the absence of preeclampsia. This a potentially important breakthrough because fetal growth restriction is the leading cause of stillbirth (worldwide, 3 million babies are lost to stillbirth).

DM 199 may reduce endothelial cell dysfunction

It was noted previously that with preeclampsia, the endothelial cells - master controllers of blood vessels - are stressed (there is endothelial dysfunction). Activation of the bradykinin 2 receptor caused by DM199 may have beneficial actions that mitigate the endothelial dysfunction and quench vascular injury beyond just blood pressure control. For instance:

1. Activation of the bradykinin 2 receptor facilitates signalling of VEGF molecule. VEGF is the important pro-angiogenic molecule contributing to blood vessel health and the creation of new blood vessels. The activation of the bradykinin 2 receptor facilitates VEGF signalling two ways. First, it relays a signal to increase activation of the VEGF 2 receptor itself that sits on the cell membrane.38 Secondly, signals are relayed into the nucleus to produce more VEGF protein as well as its main receptor (VEGF receptor 2).39 There is a large body of literature showing tissue kallikrein (the natural version of DM199) is centrally involved in angiogenesis where VEGF is likely to play an important role in this new vessel creation.39,40

2. The downstream intracellular molecules switched on by the bradykinin receptor 2 may reduce oxidative stress.41,42 Reducing oxidative stress would be advantageous as preeclampsia is associated with increased placental and systemic oxidative stress.

3. The downstream intracellular molecules switched on by the bradykinin receptor 2 may reduce inflammation. For example, nitric oxide does not just dilate blood vessels but also has inflammatory dampening actions.43

4. Stimulation of the bradykinin 2 receptor also increases insulin sensitivity, glucose uptake44 and glycogen synthesis, reducing levels of glucose in the bloodstream. High blood glucose can injure endothelial cells. Reducing blood glucose levels may further reduce endothelial dysfunction (the preeclampsia risk is higher among women with diabetes during pregnancy).

All these actions to reduce endothelial dysfunction, a hallmark of preeclampsia, may help reduce disease severity (over and above simply lowering the blood pressure).

DM 199 has been shown to be safe in humans and decreases blood pressure. Early phase first in human trials of DM 199 have shown it to be safe (see section 5). DM199 has and is being tested for chronic renal disease and stroke (see section below on DM199). Furthermore, that DM199 causes consistent falls in blood pressure in humans is reassuring. This is important 'proof of principle' data that DM199 is indeed active in humans and likely to be switching on the natural molecular circuitry, as hypothesised. It seems extremely likely it can drop blood pressure for women with preeclampsia.

Most drugs currently used to treat pregnancy conditions are also drug versions of natural molecules.

While DM 199 is a very novel approach to treat preeclampsia, it is notable most drugs used to treat obstetric conditions are drug versions of natural molecules: these drugs commandeer the body's natural molecular machinery to exert beneficial effects. For instance, natural progesterone is given in a vaginal pessary to reduce the risk of preterm birth,45 various types of prostaglandins (not prostacyclin) are given to prime the cervix in preparation for labour46 and synthetic oxytocin (a short peptide released from the pituitary gland) is given to switch on active labour.

Hence, the concept of giving a drug that is an analogue of a compound that naturally occurs is not radical.

Circulating levels of Kallikrein-1, the natural form of DM199, may be reduced in preeclampsia.

A Chinese research group reported that blood levels of kallikrein-1 in 51 women with preeclampsia were around half of levels seen in 45 pregnant women without preeclampsia. The reduction was even more acute in women with severe preeclampsia.47 Furthermore, an older study that studied 198 women reported kallikrein-1 levels in urine among women with preeclampsia were half levels seen in those free of the disease.48 DM 199 is likely to be safe in pregnancy as it is too big to cross the placenta The majority of current drugs cross the placenta. Most drugs are small molecule drugs and readily diffuse across the cell membrane of the placenta to enter the fetal circulation. While most won't cause harm to the fetus (the fetus has a liver and kidneys capable of metabolising drugs to protect itself), some might.

Drugs that are large proteins do not cross the placenta as they are too big to diffuse through the cell surface. An active transport mechanism. This makes protein drugs ideal for treating maternal conditions. Currently, there are very few protein drugs on the market.

DM 199 is a protein drug. Thus, using DM199 to treat preeclampsia is attractive because, as a protein of around 268 amino acids (the size of kallikrein-1), it is too big to passively diffuse across the placenta into the fetal circulation. This means it may be less likely to cause untoward effects on the fetus.

Summary DM199 is a promising new treatment for preeclampsia for the following reasons

1. Reduces blood pressure, a hallmark of preeclampsia

2. May reduce endothelial dysfunction and maternal blood vessel injury

3. May rescue the placental disease

4. Safe in animal and human studies

5. DM199 is too big to cross the placenta

6. Animal reproduction toxicities studies in pregnant animals suggesting there is no harm to the offspring (see section 2).

7. In a pregnant population, patients with preeclampsia may derive most benefit from DM199, justifying the cautious dose-finding of DM199 DM199 may also be a treatment for fetal growth restriction

8. The placental rescue (if the uterine arteries dilate) may mean DM199 can increase placental perfusion which will increase oxygen and nutrient deli

For these reasons, we will propose undertaking the phase I/II clinical trial outlined in this protocol.