For the remainder of today’s lecture we’re going to look at in vitro and in vivo preclinical development. We’re getting away from CMC and we’re going to be focusing on the pharmacology and toxicology studies that one needs to do in order to get into the clinic

Now, the reason that we have to do these pre-clinical studies is because subjects in clinical trials cannot be exposed to risks that have not been evaluated first in non-clinical studies. When we do our first in human study, we focus on estimation of a safe starting dose, and monitoring for adverse events that may appear during the clinical trial.

These first in human studies usually being with a single ascending dose, studied in healthy volunteers. And the goal is evaluating the pharmacokinetic parameters and drug tolerance. For our subsequent studies, or our later phase studies, phase two, phase three studies, these studies are usually performed in patients, and we increase … or the dose may be different, duration of the trials are certainly much longer, and the number of subjects are increased.

So, for the early phase studies, the trials are initially exploratory in nature, looking at safety and advocacy, and then our later phase studies confirm the data, and confirm what we’ve learned in the early studies in regard to safety and advocacy.

In order to develop the pre-clinical program, the clinical plan must be thought through beforehand. That’s because we want our pre-clinical program to be as close to the clinical program as possible, because they want to take our results from the pre-clinical phase and transition and translate them into the clinical phase.

So when we design our animal studies, we need to understand the duration of treatment for the phase of clinical development that we’re in. We need to understand the dose schedule for that phase of clinical development, and the [inaudible 00:02:10] administration, because we want those animal studies to mimic the clinical trial as close as possible.

Now, ICH has a lot of guidelines to assist us in our preclinical studies. There’s two things I’d like to point out here on this slide. All of these different guidelines are types of clinical studies that we do or are what we look for in preclinical studies prior to going into the clinic. For example, the S1 Guidance is on carcinogenicity and we need to do a study in animals investigating the cancer-causing potential of a drug before we take the drug into the clinic for long-term use. Toxicology studies also need to be done. We need to look at reproductive toxicology studies.

Now, S6 is the guideline that we use for the biotechnology-derived products and the focus of this presentation will come from the S6 document. If you would like more information, you can search for the document on the ICH website and I’d be pleased to give you that address if you need it.

Now, the first studies that we do are usually in vitro studies, and we do these studies in cell lines or primary cell cultures where we look at a biological activity or potency. We looked at receptor occupancy, receptor affinity, how the molecule binds to the receptor, et cetera. All of this has to be done prior to going into the clinical situation.

We also do these studies in order to assist us in selection of the relevant species for toxicology, and we use cell lines and receptor binding assays to do this, too. So before we go in vivo, before we enter an animal study, we do work in the laboratory using cell lines.

These studies are designed to assess qualitatively the relative sensitivity of the species to the biologic. The last thing you want to do is to do an animal study in an animal that is not sensitive to your product because then the results that you get cannot be translated to the clinical situation, and you will need to redo your studies, wasting time, wasting money, but also it’s unethical to do this.

Now, for monoclonal antibodies, we perform cross-reactivity studies to look at the immunological properties of the molecule such as antigen specificity. We look at complement binding, unintentional reactivity, and any other cytotoxicity towards human tissue. So that’s a study that’s specific to the monoclonal antibodies, and it is a study that will also help in pinpointing the relevant species.

Now, what is the definition of relevant species? This is one of the first decisions one needs to make before one begins their in vivo toxicology program. Well, a relevant species is a species in which the test material is pharmacologically active due to the expression of the receptor or an epitope.

There’s a variety of techniques that we’ve used, and we’ve already talked about these techniques. For example, you may do an immunochemical technique or some type of functional assay, and you may also do a literature search looking at homology of the animal receptor to a human receptor. As I mentioned for monoclonal antibodies, we demonstrate similar tissue cross-reactivity to human tissues.

Normally two relevant species are required for toxicology. One is a rodent, and one is a non-rodent. The rodent species are usually mice or rats. The non-rodent species are dogs or monkeys. In certain cases, only one relevant species may be available. Usually in long-term studies, two species are used that show a similar short-term toxicity. However, in some cases, only one species may be necessary. But as a general rule, two relevant species are required.

However, in some cases, there may only be one relevant species. If one can demonstrate through data that there is only one relevant species, then only one relevant species is required for toxicology.

One of the types of studies that we do as part of our [Invivo 00:00:07] preclinical program is a pharmacokinetic and toxicokinetic study. What this study does is it provides exposure data. We frequently call these add me studies. We look at absorption. We look at distribution. We look at metabolism and we look at excretion of the molecule.

Toxicokinetics is defined as the relationship between the systemic exposure of a compound and it’s toxicity. Pharmacokinetic and toxicokinetics studies assist in the comparison of toxicology within the animal species that one does the study and extrapolation of those results into the clinical situation.

These studies can contribute to setting the optimal dose escalation that when used in clinical development and they can also assist in modifying the intended dose, the route or the schedule for the clinical trial. These studies are strongly encouraged and they’re considered to be routine and inspected in all toxicology programs

Now, the toxicology studies that we do estimate the safe starting dose for the clinical studies. They look at the relationship to exposure and estimate the start escalation, dose escalation, scheme. They also assess the toxic effects, which result or with respect to target organs, allowing for monitoring of toxicity in clinical studies. We do these studies using varying dose schedules and varying doses. We also look at the ability to reverse the effect of the drug. So in that way if we are in a clinical study and we do see a toxic reaction, we need to be able to reverse that toxic reaction. These studies also allow us to assess the hazards that cannot be evaluated in human trials. For example, we cannot do teratogenicity studies in humans, it is unethical to do these type of studies in humans, so we have to do them in animals. Carcinogenicity studies in humans also cannot be done because it is unethical, so we do these studies in animals. We also can’t sample tissues in humans, looking at drug effects. So these are also done in our in vivo pre-clinical program.

Now when we do these studies and we do a low dose, we do a high dose and we do a mid dose. So there are three dose ranges that we frequently use in addition to a placebo. The high dose is usually based on a maximum tolerated dose, a dose in which we see toxicity to a certain extent. However, with some of our biologics, we don’t see any toxic effects. So if there is no observed toxicity, then the dose is based on the maximum feasible dose. For the animals that we use in these studies, they’re limited by their size to the volume that we can give them, especially with the biologics ’cause these are mostly parenteral administrated drugs, so you can only give a certain volume to an animal, and so we’re limited in that way.

Now, the low dose that we use in these studies is usually based on the No Observed Adverse Effect Level, or what we call the NOAEL level. So we do, when we design these studies, we have a placebo. A placebo is, in general, the product without the active ingredient. We’ve got the low dose, which is based on a level where we see no observed adverse experiences, and we’ve got a high dose, which is based on toxicity, it is based on a maximum tolerated dose, or it may be a maximum feasible dose, and then we’ve got a dose in between. And all of these doses are designed to simulate the clinical situation, and these studies are designed using the same route of administration and a comparative dose level to what we’re going to do within the clinical trial.

Now we do single and multiple dose toxicology studies. We start out with a single-dose study and in these studies the information that gives us assists us in describing the relationship of the dose to systemic or local toxicity. They’re also used to select doses for the repeat-dose toxicology study. We usually do two routes of administration, and this is recommended in guidance. One of the routes should be the intravenous route. The other route is usually the route in which the drug may be used in the clinical situation. For the parenteral biologics this is usually subq or IM.

The repeat-dose studies are done with various durations. The duration depends on the length of the human clinical trial that one is conducting. We need to ensure that the duration of the repeat-dose studies covers the duration of the clinical trial. The duration of these studies needs to be equal to or exceed the duration of the clinical trial. If the trial is, for example, a two week long trial then we will do a repeat-dose study that usually lasts about one month perhaps and usually do this in two species. If a clinical trial is greater than six months, then we will frequently do a six month study in a rodent and perhaps a nine month study in a non-rodent species.

Recovery periods are designed into these studies to determine if any adverse events are reversible once the drug is no longer being administered to the animal or to see if there’s any worsening of effects or any delayed toxic effects. For proteins we also monitor immunogenicity. Immunogenicity is the formation of anti-product antibodies. Immunogenicity is frequently seen in our in vivo studies because the molecules that we give to the animals are designed to bind to human receptors and they are copies of human molecules. When the animal’s immune system sees these molecules they recognize them as being foreign and they mount antibodies to them. But even though it is somewhat expected, it’s not seen in all cases and we do monitor it.

When we monitor immunogenicity we look at the effects of the antibody formation on pharmacokinetics and pharmacodynamics. We look at adverse events for the monoclonal antibodies. We look to see if complement has been activated if that’s expected. We look at any sort of new toxic effects that the anti-product antibodies may have resulted in. Now, immunogenicity in an animal model is variable. As I said, sometimes you’ll see it and sometimes you won’t which is frequently what you see in the clinical situation. In some patients you may see anti-product antibody formation. In other patients you don’t. Most of the time when you do see it it is transient and it is not linked to any clinical symptoms, although in rare cases it an result in a severe clinical response. But what we also see in the animal model is usually not predictive of what one is going to see in the clinical trial.

Now, the formation of anti-product antibodies can also confound the results of the toxicology study that one does just because it generates a lot of noise. Now, for the small molecule products it’s recommended that guinea pig anaphylactic studies be done. However, for the large molecule studies or for the protein products we don’t do these because they’re usually positive and they’re not reflective of a human response so therefore they aren’t done.

Local tolerance studies are another type of study that we do as part of our pharmacology toxicology program. For these studies we’re looking for a response in the local site of administration, so we use routes that are relevant to the proposed indication. For example, in our clinical study, if we’re going to have a sub-q indication, then we do our local tolerance study using a sub-q indication.

Now, these studies need to be performed prior to human exposure and using the marketed formulation. If you change the formulation, you will more than likely have to redo this study. What I recommend is that when one designs the toxicology program that instead of doing a separate study looking at local tolerance, that one works in local tolerance endpoints, as part of a repeat dose toxicology study instead of a separate study. That saves money. That saves time and it also saves the, well, it also prevents the use of additional animals where they may not be necessary.

All safety pharmacology studies are done to investigate the potential for physiological effects that may not be detected in standard toxicology studies. So, these are studies where we look at the effects on general systems. For example we do an evaluation of the central nervous system, the cardiovascular system, renal system, and respiratory systems. These are usually the systems that we look at.

Now, frequently for the biologics, these don’t have to be separate studies. For small molecules they are separate studies, but for the biologics what we do is we work in safety pharmacology end points and measures into the general toxicology studies, or, as I said, you may do them as separate studies, but for biologics you usually don’t.

Genotoxicity studies are done to evaluate mutations and chromosomal damage that may result from the molecule, and these studies have to be done prior to the first in human study. So prior to beginning your phase one study you need to do genotoxicity studies. And these are just a standard battery of tests. The assays that one usually does is you do an assay in bacteria to detect mutations in a target gene, and these are usually salmonella or E. Coli, what we call the Ames Test. So it’s an in vitro study that we do. We do an assay in mammalian cells to detect chromosomal damage, and these are usually done in either Cho cells, a Chinese hamster ovary cells, or in mass lymphoma cells. And we also do an in vivo study, a mouse micronucleus assay where we look at chromosomal damage to the hematopoietic cells. Now, these studies are designed to detect immunogenic effects of small molecule drugs, chemicals and environmental agents, but because proteins do not interact with DNA or chromosomal material we usually don’t do these studies for protein products.

Carcinogenicity studies are done to evaluate the potential to cause cancer, but we only do them if patients will live more than two to three years longer or the life expectancy is significantly prolonged due to treatment and the exposure will be prolonged or where there may be a cause for concern.

Now, a prolonged exposure means a continuous dosing for six months, a repeated intermittent dosing or long-term tissue retention. And cause for concern, examples of that would be if the chemical that you’re administering resembles a known carcinogen or if it induces preneoplastic lesions in your acute or repeat-dose tox studies.

So under those conditions, one would need to do carcinogenicity studies. So patients living longer than two to three years or if life expectancy is significantly prolonged due to treatment, and exposure will be prolonged or where there may be cause for a concern.

Now the timing for these studies really depends on the indication and duration of treatment but during the phases of clinical development, phase one, two, and three, usually these studies are done in phase three or later. For example, they may be done as post-approval studies.

Reproduction, a toxicity study’s evaluates fertility and early embryonic development, the effects of embryo fetal development, and the effects on late gestation to the sexual maturation of the offspring, so it evaluates early development and late development on the embryo and also evaluates fertility. Now, these studies are important in that they govern which subjects you can use in clinical trials. For example, men may be included in phase one and phase two studies prior to the conduct of a male fertility study, because when one performs the repeat dose toxicology studies, one usually evaluates the male reproductive system, and because that reproductive system has been evaluated in repeat dose toxicology studies, we use men in phase one and phase two trials. Now …

Now, the guideline for including women not of childbearing potential is similar to the inclusion of men in clinical trials. Now, the definition of a woman who is not of childbearing potential is a woman that has permanently sterilized, she’s post-menopausal. The risk of becoming pregnant is extremely low.

Now, these women may be included in clinical trials without reproductive toxicology studies provided the relevant repeat dose toxicology study has been conducted, and by relevant repeat dose toxicology study, this means that the study had to include an evaluation of the female reproductive organs.

So, prior to the inclusion of men in clinical trials, prior to the inclusion of women not of child bearing potential in a clinical trial, we need to ensure that when we design the repeat dose toxicology studies, that the male and or female reproductive organs have been evaluated. If that evaluation has been conducted, then we can include these individuals in our studies as long as the risks are low.

Now, women of childbearing potential is a different story, and that’s because of the risks to the embryo, the risks to the fetus that may be induced by this investigational drug. So we look at these studies differently. We look at the reproductive toxicology studies differently. We look at what we need to know about this investigational drug prior to including these women in our studies.

Now, under certain condition, women of childbearing potential can be included without reproductive toxicology studies, and that is if one takes the appropriate precautions to minimize risk. Now, that means that one needs to conduct pregnancy testing, and that the women must be on a highly effective birth control. And this needs to be written into the clinical protocol, and one must conduct testing, and monitor the pregnancy status during the clinical trial.

Informed consent must include any known pertinent information related to reproductive toxicology so that these women can adequately make the decision to participate or not participate in the clinical trial. Now, if one has no relevant information, then the informed consent must state the potential for risk, so one must tell these women that there is risk to an embryo, to a fetus, even if there is no relevant information.

Now, the assessment of female fertility and embryo fetal development should be completed before women of childbearing potential using birth control are enrolled in phase three studies. All female reproduction toxicology studies and geno toxicology studies must be completed prior to inclusion in clinical trials of women of childbearing potential not using highly effective birth control, or whose pregnancy status is unknown. The definition of highly effective birth control is a failure rate less than one percent per year when the birth control is used consistently and correctly.

Pre- and post-natal development should be studied prior to submitting the marketing application, after we’ve completed phase three trials and we move into the phase where we’re going to submit the BLA, we’re going to submit the NDA, then we submit pre- and post-natal development data. However, if there is cause for concern, this data needs to be submitted earlier.

So the take-home point for reproductive toxicology studies is that it depends. The type of study that one performs, and the stage of development where this is conducted depends upon the population that one wants to include in your clinical trial. If you’d like to include men, if you’d like to include women not of childbearing potential, then you need to ensure that your repeat dose toxicology studies looks at female reproductive organs, looks at male reproductive organs.

If you want to include women of childbearing potential, one needs to ensure that their pregnancy status and birth control use is monitored and tested during the clinical trials. We need to ensure that risk to an embryo, to a fetus, is minimized in these women because this is an investigational drug still. We still don’t adequately understand the risks.

Now, if you’d like to use women of childbearing potential on birth control in phase three trials, their fertility and embryo fetal development studies need to be completed before these women are used. And for pre- and post-natal development, this data needs to be submitted in the marketing application or earlier if there’s cause for concern.

So this is very much a case-by-case basis, depending upon what population you want to include in your clinical study, and what data you have generated during your reproductive toxicology study. So that’s really the bottom line when it comes to reproductive toxicology.

Next we’re going to look at a case study of a real life pharmacology/toxicology program for Biologic. But before we do this, I want to first do a recap of the preclinical presentation. The reason that we conduct preclinical studies are that clinical trial subjects cannot be exposed to risks that have not been evaluated in nonclinical studies. These risks must be mitigated and monitored during the clinical trial or they may lead one to not move into the clinical phase of development if they’re too high.

The design of the preclinical study should mimic the clinical study as close as possible in regard to duration, dose, route of administration, et cetera so one can transition from the preclinical phase into the clinical phase and also so that one can translate these data from an animal model into what one may anticipate to see or be prepared to see in a clinical situation. And that will allow us to monitor for these effects.

The first step to the initiation of a preclinical program is the selection of the relevant species. Don’t forget that the definition of a relevant species is a species in which the test material is pharmacologically active due to the expression of the receptor in the animal model or an epitope. Normally there are two relevant species, a rodent and a non-rodent species, although for some molecules they may only be one relevant species.

A preclinical program consists of a wide variety of studies. Usually it begins with a pharmacokinetic, toxicokinetic study and then moves into a single ascending dose study, a repeat dose study. Carcinogenicity studies are done. You look at local tolerance. You look at safety pharmacology. So there’s a wide variety of studies that need to be considered as part of the program. One also needs to consider when to do these studies. So there’s a tight link between the preclinical program and the clinical program. Some of the studies for the preclinical program will be conducted prior to entering phase one. Some of the studies will be conducted at the same time as one is conducting the clinical studies. It really just depends.

The preclinical program for a chemical drug is a more scripted approach. However, for a biological the approach is much more tailored to the molecule, the structure of the molecule, and the mechanism of action of that molecule. So please read Joy Cavagnaro’s article that I gave you for assigned reading because she does go through that very well. She covers the differences between a small and large molecule when it comes to the design of the preclinical program. She also goes into the preclinical program and the different types of studies very nicely. We are now going to look at a case study of a product called Aranesp. This was a product that’s manufactured by Amgen. It is a recombinant product. It is a glycoprotein, and it’s comparable to endogenous erythropoietin. Erythropoietin is a protein that we produce naturally in our bodies, and it regulates the rate of red blood cell synthesis and maintains your red blood cell level, and it’s produced in the kidney. This protein is necessary for survival. It’s necessary for growth and differentiation of your progenitor cells in your bone marrow, so it’s something that you have to have. It’s necessary for life.

Now, Aranesp is a long acing erythropoietin. Amgen does have a immediate release or a shorter acting erythropoietin called Epogen. This product was approved in 1989, and it’s given subQ and IV three times a week. Amgen then developed a follow on product called Aranesp, and what they did was they added two addition N-linked oligosaccharide change to their Epogen, and they developed this long acting product. It was approved in 2001, and instead of giving it three times a week, you now give it one time a week. While this product does have a lower potency than Epogen, it does have a longer serum half life, and it is pharmacologically active in rats and dogs. Rats and dogs are the relevant species for pharmacology and toxicology of this product.

This product is indicated for the treatment with anemia associated with chronic renal failure and inpatients with non-myeloidal malignancies, where anemia is due to the effects of concomitantly administered chemotherapy. So, since Aranesp or since the protein regulates red blood cell production, you give it to patients that have anemia. That anemia can be due to various reasons. Now, the information in this case study I obtained from a toxicologist review from the BLA, and you’ve got the reference here at the bottom of the slide. Please feel free to take a look at it, if you’d like it in more detail.

Now let’s start this with two definitions, because this will enable the remainder of the presentation. It will allow you to better understand the information I’m going to present to you.

Good laboratory practices are a set of principles that provide the framework within which laboratory studies are planned, performed, monitored, recorded, reported, and archived. GLPs help assure the regulatory authorities that the data submitted are a true reflection of the results obtained during the study, and can therefore be relied upon when making [inaudible 00:00:46] safety assessments. So the animal studies that we conduct need to be conducted according to good laboratory practice in order to be considered for approval of a drug. If a study has been conducted under non-GLPs, then that study is not considered as part of the approval package for a new drug.

Good manufacturing practices, or GMPs, are a holistic approach of regulating the manufacturing and laboratory testing environment. And this includes documentation of every step of the manufacturing process, manufacturing activities, operations, qualification of all manufacturing and testing equipment, validation of operational methods and procedures, a validation of facilities, etc.

So GMPs cover manufacturing. GLPs cover animal studies.

This is a listing of the in vivo pharmacology studies that the company conducted on Aranesp. Now, please remember that Aranesp is a long-acting form of a product that had already been on the market. Epogen had been approved in 1989. Aranesp was approved in 2001. So Epogen had been on the market for 12 years prior to Aranesp being approved. So, there was a very large body of data in patients from Epogen.

Now, let’s look at this in vivo pharmacology studies that Amgen did in order to demonstrate the comparability of this new long-acting product to the product that they already had on the market. Now, these are all comparative studies. NESP, N-E-S-P is Aranesp and rHuEPO is recombinant human erythropoietin, the product that was approved in 1989.

So, starting in 1993, Amgen began their first comparative study, looking at the effect of Aranesp compared to Epogen. And they looked at the hematocrit levels of normal mice. Hematocrit will measure the red blood cell volume in the blood, and that’s what they were measuring in mice. So, they did the first study in ’93 and they did nine studies from ’93 to ’97, looking at and comparing the effects of red blood cell volume between a long-acting form and the form that was already on the market.

Now, only one of these studies was a GLP study, and that was the study conducted in April of ’96. The rest of these studies were non-GLP. Now, why would a company want to conduct a non-GLP study, because it isn’t supportive of approval? Well, one reason a company may conduct that is because they’re cheaper and faster than doing a GLP study. So, it’s kind of … It’s like a quick and dirty way to gain data. And that’s what was done here, just some very quick non-GLP studies looking at the effect of this hyperglycosylated molecule against the molecule already on the market.

Now, this GLP lot produced in 1996 was also used in their toxicology studies. Now, they then manufactured in 1997 their first GMP lot. In order to conduct a clinical trial, in order to use data, in order to use a drug in a clinical trial, it must be manufactured according to GMP. So this last lot that we see that was manufactured in ’97 was manufactured according to GMP and then was thus used in the clinical studies.

So, after doing all of these studies, what’s the conclusion? Well, the conclusion was that there was a dose-dependent increase in hematocrit, and also that Aranesp introduced a fourfold … Was a fourfold more potent product than erythropoietin when given three times a week. And my guess as to why so many studies were conducted is that there was some remodeling going on. The glycosylated chains were being structurally changed in some way, the molecule was being remodeled and each time the molecule was being remodeled, an in vivo study was done, a comparative study, looking at the differences between the long-acting and immediate release form.

So, this is a real-life example of what an in vivo pharmacology study may look like, or program, may look like, and how one may use an animal model in order to remodel a protein to demonstrate or to give an effect that one wants to have.

The company also did a long list of pre-clinical studies. In one study, they looked at immunogenicity, as we discussed. They injected mice sub-q and IV. There was a concern that because Aranesp had a longer half life, then it may be more immunogenic, because it remained in the body for a lot longer. Maybe it was more likely to induce an anti-product antibody formation.

However, the conclusion from the study was that the antibody titers were similar. The antibody levels did not increase due to the long-lasting effects of the molecule and that the sub-q route of injection was more immunogenic than IV. That’s not unusual for a biological product. Frequently, the sub-q route is more immunogenic than the IV route, so that’s very usual.

The company also conducted in vitro pharmacology studies in cells. These studies demonstrated comparable receptor binding activities and stimulation of erythroid progenitor cells. In cells, they studied the binding of this product just to make sure that it had comparable binding and that it did bind to the expected receptor. They also looked at if the molecule stimulated these early cells, these erythroid progenitor cells, that would then go on to produce red blood cells.

Next, a long listing or a high number of pharmacokinetic ADME studies were done. Three single dose studies were done. One was done in rats, both using the IV subcu route of administration, one was done in dogs, and these were just with Aranesp and then a comparative studies was done in dogs using both Aranesp and Epogen. A multiple dose PK study was done. And then they did a PK study looking at rats that had had their kidneys removed. Some had one kidney removed. Some had both kidneys removed to see if, well I expect, to see results because this product would be used in patients who are on dialysis so patients that had renal impairment.

An elimination study was done and all of these study were done non-GLP. A distribution and excretion study was done under GLP and then a couple more non-GLP studies were done. Please note that the one study looking at HSA-containing and HSA-free lots is looking at a formulation change. So sometimes a formulation change may be tested in an animal model to look at the results. HSA is human serum albumin so the company looked at dosing of product with human serum albumin and in human serum albumin free lots and this is probably because they were considering removing HSA from this product.

Now the conclusion by the FDA reviewer from all of these studies was that Aranesp showed expected exaggerated pharmacological response consisting of increased red cell mass and a dose dependent increase.

These are the single dose toxicology studies that were done. A single dose and acute dose, more or less means the same thing here.

Two studies were done in rats, one non GOP and then one GOP study. You see the same thing with a single dose studies and acute dose studies, IV in dogs, first a non GOP study is done and then a GOP study is done.

These studies show the expected exaggerated pharmacological effects consisting of increased red cell mass and increased [reticulus 00:00:38] site levels, so they share the expected results.

The next bullet point, the last one is very important because we’re going to talk about setting the dose for first in human study. A method that can be used is to look at the KNOEL, the know observed effect level. That level is determined based on your toxicology studies.

For this study there was no observed effect level because of the exaggerated pharmacological effects of the product in the animal model. This drug was well tolerated up to 150 micrograms per kilogram in dogs and 200 micrograms per kilogram in rats.

There is a long listing of repeat dose toxicology studies. Once again we see the non GOP studies being done and then the GOP studies. A 14 day toxicology study was done first on GOP and then a four week study was done. One in rats and then another in dogs. And with both of these studies there’s a four week recovery period. And if you remember we talked about the importance of a recovery period to see if any adverse events will be minimized or to look if any toxic events will appear once dosing has stopped. And with the recovery period, the way one operationally conducts these studies, is that one stops dosing and one observes the animals. Now in the four week tox study in rats, the FDA reviewer disagreed with the company. The company said that the Noelle, the no observed adverse effect level was greater than 100 grams per kilogram per dose for both rats for ministration. However, the FDA reviewer noted that this was not achieved as exaggerated pharmacological responses were noted at all dose levels. And you see the same sort of response for the four week toxicology study in dogs, of that was done IV.

The company then did a subcu study in dogs for four weeks and then moved on to a three-month study, a six-month study, and then a 26-week study.

So as you can see, as the product went through the different phases of clinical development, or perhaps this is something that you’ll appreciate later on in the course, is that as the duration of the clinical study increases in length from phase one to phase two to phase three, then one needs to have toxicology studies completed that cover the duration of the administration of that investigational drug in the clinical situation, and that’s what you see here. You see this stepwise approach to pre-clinical development which is simultaneous to or prior to the stage of clinical development in which the study drug is used.

A separate local tolerance study was done looking at four different routes of administration in rabbits. Two other studies were done. One looking at a tissue binding to human bone marrow, liver, and pancreas, and that’s more than likely due to look for non-specific binding of this molecule to other tissues. And there was also another study done looking at human tissues in general.

Now, the third major bullet down mentions a single-dose tox study with Aranesp and Aranesp EL in rats. And this study was conducted looking at a potential impurity. So remember, back in the CMC section, we talked about impurities and the importance of identifying them, but then also qualifying the impurities and looking at the effects of those impurities in an in vivo system. And that’s what the company did here. My guess is that they separated out this impurity or they took a sample that had the impurity in it and compared it to a sample without the impurity to see what effect that would have in an animal model. So that’s one manner one can go about evaluating an impurity that has appeared in the manufacturing process. Now, after all these studies, the FDA reviewer concluded that there was no NOAEL due to the exaggerated pharmacological effects.

Now, for carcinogenicity studies, the company provided written justification to the FDA that there was no reason to conduct these studies. The justification they provided was that the immediate release form Epogen has been administered in thousands of patients for many years, so there is a large safety database on this product. There’s a lot of data on this produce. And the long-acting form has been shown to bind only to erythropoietin receptors, so it’s binding to the same receptors as the immediate release form.

In addition, while looking at a tissue panel, this receptor was detected only in the bone marrow, or was detected in the bone marrow only when looking at a human tissue panel. Therefore, there really is no evidence, in vitro evidence, to think that this product would cause cancer in a person.

In the 6-month rat and dog study, the company looked at a mycotic activity and hypoplasia, and they detected no evidence of that with exception of the bone marrow. And they also said that there are no known epo-response tumors, no neoplasms associated with renal failure. Therefore, based on this risk analysis that they’ve done, the potential for causing cancer from enhanced proliferation of erythropoietic cells due to Aranesp and Epogen administration is not there. Therefore, there was no need to conduct carcinogenicity studies.

And this is the type of justification that one frequently sees for biologics, because for biological products, one cannot do the long-term type of carcinogenicity studies that one does for small molecules. The carcinogenicity studies that are done for small molecules take place over the lifetime of the animal, and they’re usually conducted in rats which live for a couple of years.

Now, to administer a protein product repeatedly for a long period of time will usually result in immunogenicity, so it would result in the formation of anti-product antibodies. And that confounds the results and it can lead to morbidity and mortality in the animal model. So the type of carcinogenicity studies that are done for small molecules are usually not conducted for large molecules due to the formation of immunogenicity. And what the company has done here, this risk analysis, is also what one frequently sees for biologics when it comes to carcinogenicity testing.

Now, a long listing of reproduction, a toxicology studies were done on [inaudible 00:00:12], and I’m not gonna go over these one by one. I just want you to be aware of all these studies and to see that the company did a lot of studies under GOP, and also they conducted NOEL for every studies, which is going to be important when we look at how one sets the safe starting dose for the first in human study, which is a very critical decision.

And here, the reproductive toxicology studies continue. And the conclusion that the FDA reviewer made was that the expected pharmacological response was seen for Aranis and that there were no teratogenic concerns seen in any of these studies.

Now for biologics, if you remember what I said earlier? We usually don’t do mutagenicity studies because these products not interact with DNA, they do not interact with chromosomes. They bind to the cells. However, sometimes you will see companies do these studies. And this company did for a protein product. And all the studies were negative and that’s to be expected. So I’m just … I just put this in here because I wanted to give you a complete picture of the toxicology program that was conducted for this molecule.

The lecture today covered a lot of material. We covered CNC. We covered pre-clinical from an in vitro and an in vivo perspective and we looked at setting the maximum safe starting dose for our first-in-human study. Next week, we’re going to look at phase one studies, we’re going to look at clinical trials in general and get more into a clinical development. Please don’t hesitate to contact me, either by email or by phone if you have any questions. Thank you.

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