My answer is you cannot conduct an in vitro trial to replace a clinical trial. Regulatory agencies do not accept it. You need to conduct animal toxicity tests one in rodent and the other in non-rodent (dogs or non-human primates) and prove that the drug is not toxic in at least two species of animals. You also need to show that the toxicity doses have a large therapeutic margin.

In vitro models are typically used to understand the mechanism of toxicity to understand the relevance of animal toxicity to humans. But there is a lot of debate about it.

Primary hepatocytes are typically the best models.

Liver slices don't work well because, drugs do not penetrate interiors of the liver slices. If a drug is not soluble enough in the aqueous solutions, the in vitro studies will be useless.

Cell lines do not have the same drug metabolism enzymes that are present in normal human liver and are not recommended

Co-cultures are not validated model. There are still lots of issues to work before they can be used routinely. Liver contains several different cell types and the mechanisms of toxicity and the pathology of drugs varies considerably, thus using co-cultures of hepatocytes and kupffer cells cannot be applied for all hepatotoxicity mechanisms.

Hope these answer your queries

I wonder whether this drug is used for clinical trials on hepatic cancer. At th start, I maintained the primary cells as my research model, however, I replaced primary cells by cell line model in order to investigate the pharmatoxicology of drugs that are used on cancer as clinical medicine.

We have used human and animal liver cells but cultured hepatoma-derived cultured cells proved a very revarding model, both Hep3B and HepG2. Co-cultures with fibroblasts are also helpful. Cell viability and electron microscopy are among valid assesment methods

If you want to spare an animal model, you have to do most of the experiments you are mentioning, and I'm not sure if you will convince anybody else.

The problem with primary cells and tissue is the lower level of reproducibility which can lead to a lot of work. So start with three or more cell lines and primary cells in cell culture, learn sth about the toxicity in order which concentrations to use lateron. Anything that can be done to be as close as possible to the in vivo situation is a good argument, so if you can refer to slices or other kind of original tissue (one system is enough) ... just perfect.

As Albert said, it depends what you want to test. You said the drug is in trials, so if there is a known human liver toxicity you want to investigate, you need to know the relevance of rodent or other animal liver vs human liver. Then you can think about which cellular model meets the specific needs, liver slice from rat is different to the models with human liver cells in vitro.

If you are asking about developing a new drug before the first in man trial, then in vitro models will not be sufficient to show systemic safety for the humans to be treated. In vitro may help understand the interspecies differences, important metabolites and the dose response etc but selecting the wrong in vitro model will give you the wrong answers, so careful selection for the specific project is critical.

Liver cell lines are quite divergent from liver and hepatocytes. The HepaRG cell line (human) comes closest when it comes to detoxification processes. Apart from that liver slices are a very good tool for detoxification studies and have been underestimated. It is, however, quite an investment to set up the technique properly

A warm thanx to all of you sir...:)

There is a book titled "Hepatocyte Transplantation: Methods and Protocols" Edited by Dhawan, Anil ; Hughes, Robin D. Edition 1 , 2009 , HUMANA PRESS. Perhaps it is useful for you. Also, Dr. Gomez-Lechon at Hospital "La Fe", Valencia, Spain works with hepatocytes since many years ago, principally in terms of metabolic-competent hepatocyte cultures . For example: http://www.sciencedirect.com/science/article/pii/S088723330300122X

Liver slices

My answer is you cannot conduct an in vitro trial to replace a clinical trial. Regulatory agencies do not accept it. You need to conduct animal toxicity tests one in rodent and the other in non-rodent (dogs or non-human primates) and prove that the drug is not toxic in at least two species of animals. You also need to show that the toxicity doses have a large therapeutic margin.

In vitro models are typically used to understand the mechanism of toxicity to understand the relevance of animal toxicity to humans. But there is a lot of debate about it.

Primary hepatocytes are typically the best models.

Liver slices don't work well because, drugs do not penetrate interiors of the liver slices. If a drug is not soluble enough in the aqueous solutions, the in vitro studies will be useless.

Cell lines do not have the same drug metabolism enzymes that are present in normal human liver and are not recommended

Co-cultures are not validated model. There are still lots of issues to work before they can be used routinely. Liver contains several different cell types and the mechanisms of toxicity and the pathology of drugs varies considerably, thus using co-cultures of hepatocytes and kupffer cells cannot be applied for all hepatotoxicity mechanisms.

Hope these answer your queries

you can also consider the humanized chimeric mouse, with a human liver, the uPA scid is the "historic" model in the field but more recently the FRG mouse appeared, from Yecuris in the US, with a high level of performance

Albert, Hauke and David have made good points that will surely help you. I would like to add that we are talking here on the closest to in vivo, which does not necessary mean closer enough to the effects you would have obtained if you are using an animal model. No matter how close these in vitro tests could be, they will never be close enough to mimic the effect in a true life situation as obtained with in vivo test. Moreover they will allow you having only a predictive acute toxicity profile. Sub acute or long term toxicity profile will still need to be accessed before true clinical trials. In a true life experiment, other body metabolites will interact with your test compound; the immune system will have a role to play, the absorption which in some cases may be dose-dependent will also interfere, other metabolites resulting from the bio-degradation of your test compound my also increase the toxicity effects. I will join Gabino to recommend to go through the book "Hepatocyte Transplantation: Methods and Protocols" for further details. We are still a little bit far from replacing animal use in testing hepatic toxicity

Thanx sir...

you can try out organotypic liver slice culture and monitoring the albumin in the supernatant and also histomorphological analysis.

pranab K das

You need to verify the toxicity in one or more animal models. In vitro assays with liver cells or slices only provide some indications or have a quick screening.

Ok sir, i got it. Thanx.

Are you familiar with HemoShear? They are doing some very interesting work, taking in vitro to a whole new level. Take a look:

http://www.hemoshear.com/what-do-we-do/

You may use isolated primary hepatocytes as a testing tool, but they will not exactly reflect the metabolism and toxicity of xenobiotics when they are tested in animal models.

Dear Ashok

You are correct to look to human biology-based models prior to testing a new drug in humans. As the proportionately high failure rate of new drugs due to toxicity problems (particularly with respect to liver toxicity) shows, animal models, although required for regulatory purposes, are not human models.

All of the in vitro/ex vivo models you mention have particular merits, depending on the stage of development you have reached, and therefore, how many therapeutics you intend to test (with implications for time & cost commitment). Some, such as liver slices (or even perfused liver segments), while not high throughput, have tremendous value in that they are human, with human-specific metabolism, and maintain native cell-cell, cell-extracellular matrix interactions. Human tissues/primary hepatocytes may not always be the cheapest option, but what price clinical failure?

If we can be of any assistance in sourcing human tissues or in vitro assays for you, please do not hesitate to get in touch at www.humanfocusedtesting.com or feel free to message me directly.

With best wishes for the new year,

Margaret

Margaret Clotworthy PhD