Recombinant KLK1: the next step in stroke and preeclampsia treatment

DiaMedica Therapeutics is a clinical-stage biopharmaceutical company driven to develop treatments for serious ischemic and vascular diseases. Central to their work is lead drug candidate, DM199, a recombinant form of the human tissue kallikrein-1 (rhKLK1) protein. 

As Chief Executive Officer of DiaMedica for more than a decade, Rick Pauls discusses how restoring KLK1 levels could improve the body’s regulation of blood flow, inflammation control and vascular health. This approach could go a long way toward improving conditions with limited treatment options, such as stroke, preeclampsia and chronic kidney disease. 

A critical protein for vascular function 

Pauls describes KLK1 as a protein essential to human health, often found at low levels in patients with vascular conditions. It plays a vital role in maintaining vascular tone and endothelial function. “It’s a critical protein,” he explains. “It has been reported that levels of this protein are low in patients who have diseases like hypertension, stroke, preeclampsia and kidney dysfunction.” 

Produced mainly in the kidneys, KLK1 acts through a cascade that results in the release of three key molecules: nitric oxide, prostacyclin and endothelial-derived hyperpolarising factors (EDHFs). These substances work together to dilate blood vessels and enhance microvascular circulation. This mechanism makes KLK1 a powerful vasodilator and potentially transformative in conditions where tissues are starved of blood and oxygen. 

“In these diseases, there’s not enough blood flow – for stroke it’s the brain, for preeclampsia it’s the placenta,” Pauls explains. “So, improving that circulation is absolutely key.” 

Restoring blood flow in stroke and preeclampsia 

DiaMedica’s research focuses on two very different, although linked, conditions: acute ischaemic stroke and preeclampsia. 

In stroke, the problem is immediate and severe: a blockage in a blood vessel cuts off oxygen to part of the brain. Following a stroke, the body naturally upregulates bradykinin 2 receptors in the region surrounding the blocked artery – the ‘penumbra’ – but lacks sufficient KLK1 to activate them. “We give DM199 systemically and because those receptors are upregulated in the penumbra, it focally vasodilates in that area, pulling in blood flow from other parts of the body,” said Pauls. 

In essence, DM199 could work alongside existing treatments such as tissue plasminogen activator (TPA) and mechanical thrombectomy, both of which physically remove or dissolve clots. “Stroke is a disease of lack of blood flow,” Pauls notes. “TPA dissolves the clot, blood flow gets re-established; mechanical thrombectomy pulls the clot out. We think this could be the next modality.”  

In preeclampsia, the issue develops slowly, as blood flow to the placenta becomes insufficient during pregnancy. “After about 20 to 24 weeks, the demands of the baby are greater than the supply of blood flow,” Pauls explains. The spiral arteries that should widen to accommodate this increased need instead remain narrow and constricted.  

“We think our drug can actually increase placental perfusion while also controlling blood pressure, because about half of these mothers have to deliver early as their blood pressure gets out of control,” Pauls said. 

Though the conditions differ, both share a common underlying feature: impaired vascular function. This suggests the drug could play an important role in treating both.   

From concept to clinic: building confidence in DM199 

Developing DM199 into a clinically viable therapy was far from straightforward. DiaMedica’s early work focused on proving that its recombinant KLK1 could reliably generate bradykinin – the molecule that activates the downstream vasodilatory effects. Thus, it was crucial that DiaMedica’s drug could produce KLK1 in a dose-dependent manner that produces bradykinin. 

This achievement was particularly significant given the number of past failures. “At least five companies over the years have tried to make a recombinant form,” Pauls says. “When we first tried to manufacture this, we followed a patent from Amgen and made it – but there was no activity.” 

Persistence eventually paid off and after years of experimentation and collaboration with several manufacturing partners, DiaMedica succeeded in producing an active KLK1 protein.  

As Pauls explains, the interesting thing about KLK1 was that it was not an untested molecule. “Bayer discovered this protein back in the 1940s in Japan,” Pauls notes. “They started commercialising tissue kallikrein isolated from pig pancreas for hypertension. Today, around 300,000 patients a year are treated with the porcine form in Japan and several companies in China also market it.” 

A human urinary-derived version of KLK1, which is chemically identical to the body’s own protein, is also used in China to treat acute ischemic stroke, with around one million patients treated each year. 

These prior uses gave DiaMedica confidence as it advanced its recombinant version. “For us, it was a different approach, we leveraged our understanding of these crude forms being used in Asia,” said Pauls. 

Preclinical testing further confirmed that DM199 closely mimics the behaviour of natural KLK1. A key step was showing that it has enzymatic activity comparable to the human urinary form, which in turn allowed DiaMedica to align the pharmacokinetic profile in patients with that reference. 

Activating the body’s repair pathway 

At the heart of DM199’s potential is its ability to activate the bradykinin 2 receptor pathway – a key regulator of vascular health. This taps into the body’s own repair system by targeting this pathway. 

Unlike drugs that simply lower blood pressure or relax smooth muscle, DM199 works by enhancing the natural balance of endothelial function. “By releasing nitric oxide, prostacyclin and endothelial-derived hyperpolarising factors, this is a very potent vasodilator that enhances endothelial integrity, reduces inflammation, thrombosis and improves microvascular perfusion,” Pauls says. 

Improving endothelial health could enable disease-modifying effects across multiple vascular conditions. 

Overcoming technical challenges 

While the biology was compelling, manufacturing an active recombinant KLK1 proved to be one of the greatest challenges in the company’s history. Pauls recalls that initial attempts based on an Amgen patent from 1989 produced an inactive protein. “We went to four or five different vendors,” he says. “It really wasn’t until we started playing around with the glycosylation – how the sugars are attached – that we found the key.” 

“By accident, maybe with a little good luck, we found a configuration of close to 50/50 high and low glycoforms,” he explains. “That turned out to be critical for activity, without that specific glycosylation pattern, the protein simply didn’t work.” 

The discovery became the foundation for DiaMedica’s composition-of-matter patents, securing intellectual property protection for the active form of the molecule. Manufacturing efficiency presented another challenge, but the company now works with Catalent, using its GPEx cell line technology to achieve high, cost-effective yields.  

Lessons in perseverance and future directions 

Reflecting on the company’s journey, Pauls says the most valuable lesson was persistence. “We tried to make it, it didn’t work. We tried again and again. It would’ve been easy to stop and say, ‘let’s do something else,’ but we knew this protein worked.” 

That perseverance – backed by evidence from both porcine and urinary forms of KLK1 – kept the team motivated through setbacks. “We just kept going, finding new vendors and new approaches, that was the critical piece,” Pauls says. 

Even with promising late-stage trials under way, Pauls sees plenty of scientific questions still to explore. “What’s unique about this protein is that there have literally been thousands of papers published on tissue kallikrein over decades and millions of patients have been treated with the porcine and human urinary forms,” he said. 

DiaMedica’s research is now diving deeper into the less-understood aspects of KLK1 biology, such as the role of endothelial-derived hyperpolarising factors, an area that remains poorly characterised.  

The company plans to continue its mechanistic studies and publish additional findings, but for now its main focus remains on its stroke trial and ongoing Phase II preeclampsia programme.  

Rediscovering an overlooked science 

For Pauls, one of the most compelling aspects of DiaMedica’s research is how it blends old and new science to create something truly exciting. 

“These two forms of the protein have been used in Asia – literally millions of times, maybe 10 million plus patients treated over the years,” he says. “It’s been considered old science; it’s been around a long time and we’re not aware of many recent attempts to manufacture it.” 

By successfully creating a recombinant form and demonstrating its activity, DiaMedica has brought this ‘old science’ back into modern biotechnology. “Naturally occurring protein levels are low,” Pauls concludes. “It’s just a critical protein and we’re looking forward to getting the next stage of clinical trials completed.” 

Meet the expert:

Rick Pauls, President and Chief Executive Officer, DiaMedica

Rick Pauls was appointed our President and Chief Executive Officer in 2009.  Mr Pauls has served as a member of our board of directors since 2005 and as the Chairman of the Board from 2008 to 2014.

Before joining DiaMedica, Mr Pauls was the Co-Founder and Managing Director of CentreStone Ventures, an early-stage life sciences venture capital fund, from 2002 until 2010. Mr. Pauls was an analyst for Centara Corporation, an early-stage venture capital fund, from 2000 until 2002.  From 1997 until 1999, Mr. Pauls worked for General Motors Acceptation Corporation specializing in asset-backed securitisation and structured finance.  Additionally, Mr. Pauls has served on the board of directors of several public and private companies.