The pre-clinical phase of development for non-alcoholic steatohepatitis (NASH) drugs faces many challenges. Biopharmaceutical companies have several options for rodent models, but they must weigh factors such as customization versus speed before deciding on the best approach.
Some of the challenges include:
- Diet: There is no prevailing wisdom in the field suggesting that one induction diet is superior to another.
- Duration of disease induction: Depending on the type of diet, it will take 6 to 9 months for models to exhibit NASH-like features.
- Translation: Novel biomarkers used in human clinical trials need further validation in rodent models.
Despite these issues, companies can reduce their risk by using short-term models for early screening. As the models move through three stages of the disease, an array of biomarkers can be measured to assess factors such as fat accumulation and fibrosis.
Customized disease models
The induction diets used for pre-clinical NASH studies are supplemented with high fat. Diets rich in nutrients such as saturated fatty acids, trans fatty acids, fructose or sucrose, or even the absence of other nutrients such as methionine and choline, may disrupt different pathways to yield NASH. At this point, different administration times and diets produce different results.
As companies begin pre-clinical trials, they may have to choose between customization and speed. Some contract research organizations (CROs) provide models that have already been induced, which reduces the waiting period. However, the client cannot specify the exact induction diet.
Alternatively, companies can request that a CRO custom-make the models with a certain diet. This approach, while more predictable, requires more time. But early screening data can be obtained through short-term models in which agents such as carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN) are used to induce liver fibrosis. These models can help companies evaluate whether their compound has anti-fibrotic features.
Three phases of disease
The disease created by diet induction progresses through three distinct phases:
(1) Accumulation of fat (non-alcoholic fatty liver disease, NAFLD). Typically, a section of the liver is examined under a microscope to confirm the disease state. Biochemical assays can indicate triglyceride levels. Lipid spectroscopy using MRI also can provide data on lipid accumulation, but this technique is specialized and can be cost prohibitive.
(2) NAFLD progresses to NASH. Typical biomarkers at this stage include elevated liver enzymes such as alanine aminotransferase (ALT). The marker cytokeratin-18 (CK-18), which is linked with necrosis and apoptosis in the liver, is sensitive and specific for increased inflammation in livers previously filled with fat.
(3) Fibrosis. When normal liver cells die, the organ responds by producing fibrotic tissue. A fibrosis index integrates levels of hyaluronic acid, tissue inhibitor of metalloproteinase 1 (TIMP-1) and amino-terminal propeptide of type III procollagen (PIIINP). A pathologist may also examine a section of the liver under a microscope to assess the amount of fibrosis.
As the disease progresses, circulatory pressure increases in the liver due to inflammatory and fibrotic changes. Blood flow in small blood vessels is blocked and portal pressure rises. Venous pressure measurements can therefore act as another disease marker.
NAFLD develops slowly in humans, usually taking decades to evolve and progress. However, rodents have a very short lifespan, and disease progression may take only several weeks or months in the experimental system. Although the rate of progression is faster in rodent models than in humans, many of the typical histological features in diet-induced rats with non-alcoholic liver disease closely resemble those seen in humans.
From pre-clinical to clinical
Many of these biomarkers, such as CK-18 and hyaluronic acid, can be aligned with endpoints in human clinical trials. For instance, patient blood samples can be tested for CK-18 and fibrosis markers. MRI scans of the liver and portal pressure measurements also can be performed.