Tailor-made solutions for preclinical efficacy research

Pharmatest Services Ltd is a rapidly growing CRO specialized in bone biology. The services of Pharmatest are designed for preclinical testing of drug candidates for osteoporosis and other bone disorders, such as cancer bone metastases. If your company is developing new drugs with bone indications, Pharmatest is the perfect partner for you.

Like many highly specialized CROs, Pharmatest started originally in October 1999 as a spin-off company from the University of Turku, Finland. Founders of the company include scientists with decades of experience in bone biology. These scientists had a long experience in developing and using models useful for preclinical testing of osteoporosis drug candidates. At first Pharmatest’s service portfolio consisted of models that had been developed by the key persons of the company during many years of intensive academic research. However, it soon became clear that in order to best serve its customers, Pharmatest had to establish itself as a fully independent company and further develop the “academic” models for commercial purposes. Therefore, in 2002 Pharmatest moved to its own state-of-the-art premises and continued to further develop its services to better suit commercial purposes and specific needs of pharmaceutical industry. For this purpose, Pharmatest obtained substantial funding from the Finnish Funding Agency for Technology and Innovation (TEKES). Since R&D is an important part of Pharmatest’s strategy, Pharmatest is constantly developing new models and improving its existing models. At Pharmatest we feel that it is extremely important to offer our customers test models that are constantly up-to-date and based on most recent scientific knowledge in the field. Staying in the frontline of bone research and producing high class scientific publications is an extremely important part of Pharmatest’s business strategy. Pharmatest constantly works towards publishing the results of internal R&D projects and projects performed for our customers in highly ranked scientific journals.

Pharmatest’s home town Turku contains the leading biotechnology centre in Finland, known as BioTurku, comprising about 80 biotechnology companies. In addition, the area of Turku contains three universities and two polytechnic schools. These all create a perfect environment for the growth of Pharmatest. Highly skilled personnel is perhaps the most important asset for a research organization such as Pharmatest. Pharmatest’s personnel strategy is built on close collaboration with the local universities and polytechnic schools. Pharmatest’s original key persons include scientists who have completed their Ph.D. studies on bone biology during their academic career before joining Pharmatest. During their academic career, they were involved in developing the test models that Pharmatest offers. These scientists now form Pharmatest’s knowledge base. Our goal now is to train our next generation of skilled scientists from students that have finished their basic studies in the university. Ideally, these students have been trained to use the test models of Pharmatest in the university already before they join Pharmatest. Thus, when they become employed by Pharmatest they are already experts in bone biology and in the test models offered by Pharmatest. Our laboratory personnel and technical staff are trained through a close collaboration with the polytechnic school, where students of the polytechnic perform their training period in Pharmatest, and at the same time learn basic skills in performing the laboratory work associated in Pharmatest’s models. After the students complete their studies they can be conveniently employed by Pharmatest with initial experience in Pharmatest’s test models. All this eventually translates into highly skilled, motivated and fast work force, which in turn means more value for our customers.

Pharmatest’s currently available testing services include various in vitro bone cell assays and in vivo osteoporosis animal models. These services can be used for preclinical testing of drug candidates for osteoporosis and related bone disorders. In addition to these services, the R&D department of Pharmatest is developing new models useful for preclinical testing of drug candidates for cancer and osteoarthritis. Pharmatest’s goal is that the panel of testing services in each disease indication will contain a comprehensive package of disease models from in vitro cell culture models to in vivo animal models. Pharmatest focuses on fast, clinically relevant, reliable and cost-effective strategies. Within this strategy Pharmatest would be able to use the in vitro cell culture models for identifying best compounds from a large set of compounds quickly and cost-effectively, and then continue the testing of these compounds in the animal models.

Bone is a metabolically active tissue where old bone is constantly removed by bone resorbing cells called osteoclasts, followed by formation of new bone by bone forming cells called osteoblasts. This event, called bone turnover, is normally in balance so that the amount of bone resorbed by osteoclasts is exactly the same as the amount of bone formed by osteoblasts. In osteoporosis, this balance is disturbed so that more bone is resorbed than is formed, leading to bone loss and increased fracture risk. Bone loss can be caused by either increased bone resorption or decreased bone formation. Similarly, osteoporosis can be treated by anti-catabolic compounds that inhibit bone resorption or by anabolic compounds that increase bone formation. The currently available in vitro bone cell culture models of Pharmatest include osteoclast assays for testing anti-catabolic compounds and osteoblastic assays for testing anabolic compounds. While these assays are specifically designed for testing osteoporosis drug compounds, they are also extremely useful for testing new cancer drug compounds, especially if the compounds are targeting bone cells or bone cell interactions. The bone cell assays of Pharmatest are performed in 96-well plates, where each well contains the same amount of cells. The cells are cultured for a desired period of time in the presence of the test compounds. Each plate contains a so-called baseline group without any added test compounds, and a control group including a well-known reference compound that gives the desired effect in the test system. In osteoclast assays, the reference compound decreases bone resorption, and in osteoblast assays the reference compound increases bone formation. The results of the control group and each test group are compared with the results of the baseline group to determine if the test compounds show the desired effect in the test system. If the results of the control group do not show the desired, statistically significant effects in the test system, Pharmatest will repeat the whole testing without additional costs to the customer until a significant effect is observed.

In the following chapter we describe an example of how our customers could optimally benefit from using our in vitro bone cell assays to identify most interesting compounds from a large set of compounds. With the in vitro bone cell assays, we offer three different project types: 1) Rapid screening projects, where one test concentration of each test compound is tested in 4 replicates; 2) Preliminary projects, where 3 test concentrations of each test compound are tested in 6 replicates; 3) Extensive projects, where 7 test concentrations of each test compound are tested in 8 replicates. Pharmatest strongly recommends using the results of a rapid screening project only to select most interesting compounds for further testing in preliminary or extensive projects, and the results of a preliminary project to select most interesting compounds for further testing in an extensive project. Finally, the results of the extensive project can be used for calculation of IC50 values and selecting compounds for further testing in the osteoporosis animal models. For example, if the customer has 100 compounds, we suggest starting with the rapid screening study. Based on the results, the customer could select 30 most interesting compounds for further testing in a preliminary study, and then select 10 most interesting compounds for further testing in an extensive study. Finally, 6 compounds could be selected for further testing in the osteoporosis animal models.

The osteoporosis animal models currently available in Pharmatest include rat ovariectomy (OVX), orchidectomy (ORX) and immobilization models and an intact mouse model of glucocorticoid-induced osteoporosis (GIO). Of these, the rat OVX model is most important because FDA requires in their guidelines that any therapeutic agent for postmenopausal osteoporosis must be tested in this model to obtain FDA approval. Ovariectomized rats develop osteoporosis rapidly after the operation due to estrogen withdrawal, and the model resembles postmenopausal osteoporosis. The ORX model is a corresponding model for males, where osteoporosis is caused by testosterone withdrawal. Immobilization causes osteoporosis to rats with a different mechanism, due to decreased bone formation, and the rat immobilization model is a useful additional test model especially for anabolic compounds. Also, the use of glucocorticoids causes osteoporosis with a unique mechanism affecting primarily bone formation. The GIO model can be used either to test the effects of new glucocorticoids with potentially less severe side effects on bone, or to test the effects of new test compounds on glucocorticoid-induced osteoporosis.

Pharmatest has access to a large number of different bone analysis techniques that are included in the osteoporosis animal models. These techniques can also be used as stand-alone services where the customer sends us samples for analysis. The techniques required by FDA that must be included in a regulatory OVX study include bone mineral density (BMD) measurements, biomechanical testing, histomorphometric analysis, ash weight analysis and measurement of biochemical markers of bone turnover. It is recommended to start testing in animal models with the rat OVX model, where we offer three different project types: 1) Rapid screening projects; 2) Preliminary projects; 3) Extensive projects. The rapid screening projects are 2-week studies that include only measurement of biochemical markers of bone turnover. Changes in bone turnover are seen much more rapidly than changes in BMD, and short-term changes in bone turnover markers have been shown to be useful for predicting long-term changes in BMD. The rapid screening projects can conveniently include several doses of several compounds, and they can be used to find best compounds and their most effective doses. This information can be used to select 2-3 doses of 1-2 compounds to be tested in a preliminary project. The preliminary project is a 2-month study including BMD measurements, biomechanical testing, histomorphometric analysis, ash weight analysis and bone turnover markers. Ideally, the preliminary testing should be first performed with young (3-6 months old) animals, and if excellent bone effects are obtained then repeated with old (12 months old) animals. When the best compound and its optimal dose have been identified and shown to have excellent beneficial bone effects in these preliminary studies, a 12-month extensive study can be performed. At the same time, anabolic compounds can be tested further in the immobilization and GIO models. The study setup in Pharmatest’s extensive OVX study has been designed according to the regulatory guidelines of FDA, and the results of this study can be used to obtain FDA approval for the tested drug candidate. Table 1 summarizes our example of an optimally designed way of using Pharmatest’s services starting from 100 test compounds.

Table 1: An example of optimizing the testing of 100 test compounds with Pharmatest’s services

Test system	Number of compounds
Bone cell assays Rapid screening project Preliminary project Extensive project	100 30 10
Animal models Rapid screening project Preliminary project Extensive project	6 2 1

For a pharmaceutical company, an optimal way of performing preclinical services for testing osteoporosis drug candidates would be to outsource them to a reliable professional CRO with expertise in bone biology. This would save a substantial amount of time and money, two extremely important elements in drug discovery. The number of CRO’s offering preclinical services for testing osteoporosis drug candidates has been very limited, and Pharmatest was founded to serve this function. Our mission has been and will be to create an outstanding panel of preclinical testing services and optimize their use to maximize the savings of time and money to the pharmaceutical industry. If your company is developing therapeutic agents with bone indications, we would be proud to be at your service.