Lupron URO
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Lupron URO - Scientific Information

Manufacture: AbbVie
Country: Canada
Condition: Endometriosis, Hirsutism, Prostate Cancer, Uterine Fibroids
Class: Gonadotropin releasing hormones, Hormones/antineoplastics
Form: Liquid solution, Subcutaneous (SC)
Ingredients: leuprolide acetate, benzyl alcohol, sodium chloride, and sterile water for injection. Each vial also contains sodium hydroxide and/or acetic acid

Pharmaceutical information

Proper name: leuprolide acetate
Chemical name: 5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-Leucyl-L-leucyl-L-arginyl-N-ethyl-L-prolinamide acetate.

or: des-Glycine10, [D-Leucine6) LH-RH ethylamide acetate.

or: [D-Leu6, des-Gly-NH210, Proethylamide9] GnRH.
Molecular formula and molecular mass: C59H84N16O12·C2H4O2      1209.41 as free base
Structural formula:
Physicochemical properties: Leuprolide acetate is a fine or fluffy, white to off-white powder, very soluble in water, ethanol and propylene glycol; pKa = 9.6.

Clinical trials

Study Demographics and Trial Design

LUPRON

Table 1. Summary of Patient Demographics for Clinical Trials in Prostatic Cancer
Study# Trial design Dosage, route of administration and duration Study subjects (n=number) Mean age (Range)
I* Phase 2, open-label,
multicenter study
LUPRON 1 mg or 10 mg
once daily
Subcutaneous
18 to 80 weeks
118
(111 had stage D2 disease)
∼66 years (42-93 years)
II Open-label multicenter study LUPRON
or
DES (diethylstilbesterol) 1 mg three times daily
202
В (93 had stage D2 disease)
--
* Retrospective control for this study was obtained from the National Prostatic Cancer Project (NPCP), Protocol No. 1300 which consisted of two treatment arms: DES (diethylstilbesterol) or orchiectomy.
Retrospective comparison of the results of Study I carried out by the NPCP. Patients received either DES or orchiectomy.

LUPRON DEPOT

Table 2. Summary of Patient Demographics for Clinical Trials with LUPRON DEPOT in Prostatic Cancer
Study # Trial design Dosage, route of administration and duration Study subjects
(n=number)
Mean age (Range)
III Phase III, open-label,
multicenter study
7.5 mg LUPRON DEPOT
injected every 12 weeks
Intramuscular
24 weeks
53 --
IV Phase III, open-label,
multicenter study
22.5 mg LUPRON
DEPOT injected every 12
weeks
Intramuscular
Minimum 24 weeks
61* 71 years
(53 to 86 years)
V Phase III, open-label,
multicenter study
22.5 mg LUPRON
DEPOT injected every 12
weeks
Intramuscular
Minimum 24 weeks
33* 69 years
(55 to 82 years)
VI Phase III, open-label,
multicenter study
30.0 mg LUPRON
DEPOT injected every 16
weeks
Intramuscular
Minimum 32 weeks
49 70 years
(54 to 84 years)
* Two patients (one from each study) were excluded from the efficacy analysis. Hence a total of 94 patients were studied.

Study Results

LUPRON

Two controlled multicenter studies were conducted to evaluate the safety, efficacy, and endocrine effects of leuprolide acetate in advanced prostatic cancer patients (Stage D2).

A further objective was to compare the efficacy of leuprolide acetate with that of DES (diethylstilbestrol).

Study I

The first study was an open study with 118 patients randomly assigned to receive either 1 mg or 10 mg doses of leuprolide acetate. Retrospective control for this study was obtained from the National Prostatic Cancer Project (NPCP), Protocol No. 1300 which consisted of two treatment arms: DES or orchiectomy.

Objective Response

For evaluation, patients were divided in three groups by prior treatment as shown below, and the NPCP criterion was used to assess the response.

Evaluable including D1 Evaluable,
N = 100 patients
Evaluable, D2 only
No Progression
Estimated Median Time
for First Progression
Group 1 = previously untreated 72% 76 weeks
Group 2 = previously hormone-treated 48% 49 weeks
Group 3 = orchiectomized 23% 43 weeks

A summary of survival for this study is presented in Table 3 below:

Table 3. Summary of Survival for Study I (N=47)
Week of Follow-Up Dead Alive Censored*
30
60
90
120
150
180
210
240
270
300
330
360
390
After Last Data:
(Week 395)
1
5
12
22
29
31
32
33
35
36
37
38
38
38
46
42
34
24
17
15
14
12
9
8
5
4
1
0
0
0
1
1
1
1
1
2
3
3
5
5
8
9
* "Censored" includes patients who was lost to follow-up.

The median survival is estimated as 121 weeks (± 6 to 10 weeks as standard errors).

Subjective Response

Bone pain: Of the 94 evaluable stage D2 patients, 26 reported no bone pain throughout the study. Of the remaining 68 patients, only one (1%) reported worsening of bone pain while 55 (81%) reported improvement, and 12 patients (18%) reported no change.

Nine patients reported normal performance status throughout the study. Of the remaining 85 patients, 44 (52%) improved, 34 (40%) reported no change, and only 7 (8%) worsened.

Dose-response Relationship

Orchiectomized patients who received 10 mg/day showed a somewhat higher subjective response rate than those receiving 1 mg/day; however, the difference was not statistically significant. Furthermore, the suppression of testosterone level was similar in the two-dose groups.

Endocrine Evaluation

Plasma levels of FSH and LH increased markedly within four hours of the first dose of leuprolide acetate in all three treatment groups. However, from Day 8 and on, FSH and LH levels had decreased significantly for all three groups.

Testosterone (T) and dihydrotestosterone (DHT) followed a similar pattern. By Day 4, T and DHT had increased markedly in both the previously untreated and hormone-treated groups, but subsequently declined to minimal levels by Week 2 and continued at those levels (identical to the minimal testosterone levels of the orchiectomized patients) for the duration of the treatment.

Safety

The most common side effects reported were hot flashes (41%), and sexual dysfunction (14%) with decrease in libido and impotence. Cardiovascular-related effects were noted in few patients. Three out of four patients had cardiovascular disease at pre-study. None of the cardiovascular events were reported as drug-related. Relationship to therapy is unknown.

This study showed leuprolide acetate to be a safe and effective drug for the treatment of advanced prostatic cancer.

Previously untreated patients achieved a better response than previously treated patients.

Study II

Study II was an open multicenter study with 202 previously untreated patients with Stage D2 prostatic adenocarcinoma.

Patients were centrally randomized to receive either leuprolide acetate or diethylstilbestrol (DES); those with definite evidence of progression or intolerable side effects on their initial treatment were crossed-over to the other treatment.

Ninety-two (92) patients randomized to leuprolide acetate, and 94 patients randomized to DES were evaluated.

Objective Response

An overall favourable objective response to treatment (No Progression) occurred in 86% of the evaluable patients on leuprolide acetate and 85% of the evaluable patients on DES.

There was no significant difference between the two treatment groups in time to first progression or time to treatment failure.

Time to first progression was analyzed for evaluable patients who had a best response of "no progression". The following are the estimated quartiles (in weeks):

Group 25th Median 75th
Leuprolide acetate 75 60 43
DES -- 61 42

Treatment failure was defined as time to first progression or to termination of study because of an adverse reaction. The following are the estimated quartiles (in weeks):

Group 25th Median 75th
Leuprolide acetate 67 49 36
DES 70 48 25

The summary of survival for leuprolide acetate and DES is presented in Table 4.

Table 4. Summary of Survival in Study II
Week of
Follow-up
Leuprolide acetate (N=94) DES (N=99)
Dead Alive Censored* Dead Alive Censored*
30 3 90 1 11 87 1
60 14 76 4 25 72 2
90 29 60 5 33 64 2
120 42 46 6 40 56 3
150 50 36 8 53 42 4
180 56 29 9 58 36 5
210 62 22 10 60 34 5
240 68 15 11 65 28 6
270 70 13 11 67 25 7
300 72 11 11 69 23 7
330 73 10 11 70 21 8
360 73 4 17 74 10 15
390 74 0 20 76 0 23
After Last
Death
Time of Last
Death (weeks)
(358) (364)
* "Censored" includes patients who were lost to follow-up prior to the number of weeks shown, or who are alive but have not yet completed that number of weeks.
Subjective Response

Patients from both groups had a significant reduction in bone pain and in use of analgesics. There was no difference in overall subjective response, performance status, urinary symptoms, or mood changes in patients from both groups.

Endocrine Evaluation

By week four, testosterone and dihydrotestosterone from both groups reached castrate levels and remained there for the duration of the study.

Safety

During the first treatment period, the percentages of patients who experienced side effects differed significantly between the DES and the leuprolide acetate groups. The incidence is presented in Table 5.

Table 5. Incidence (%) of Adverse Reactions During First Treatment Period (199 patients)
DES
(N=101)
Leuprolide acetate
(N=98)
Hot flashes
Peripheral edema
Nausea/vomiting
Impotence
Gynecomastia (Breast pain)
Bone Pain
Musculoskeletal spasms
12
31
19
14
63
2
9
55
15
8
4
8
5
1

Since prostatic cancer patients are already at high risk for developing thromboembolic and other cardiovascular diseases because of age and existing malignancy, leuprolide acetate offers an important alternative to treatment with estrogens.

Efficacy

Eighty-six percent (86%) of the patients on leuprolide acetate and 85% of the patients on DES had a favorable response to treatment.

In summary, this study showed that leuprolide acetate is a safe and effective treatment of Stage D2 prostatic cancer.

LUPRON DEPOT

Study III

A Phase 3, open, multicenter study was carried out to determine whether the LUPRON DEPOT 7.5 mg (1-Month SR) injected intramuscularly once every four weeks would reduce testosterone to, and maintain it at, castrate levels (≤ 50 ng/dL) in 56 previously untreated prostate cancer patients, and to evaluate objective clinical response.

The results are as follows:

Objective response

Best objective response was determined over a period of 24 weeks for 53 evaluable patients. Eighty one percent (81%) of the patients responded favourable (no progression) to treatment at some time. This result was not significantly different from the response rate of 86% observed for patients receiving the daily subcutaneous injection of leuprolide acetate solution reported in the previous study.

Testosterone levels

The median time to onset of castrate levels of testosterone for 53 evaluable patients was 21 days, and mean testosterone levels fell within the castrate range by Week 3 of treatment. After the onset of castrate levels, there were no escapes of testosterone values, provided that patients received their monthly injections on time. The pattern of testosterone release over the first

24 weeks of treatment did not differ from that observed in patients receiving the daily subcutaneous injection of leuprolide acetate solutions when an injection was delayed by 7 to 12 days; testosterone levels remained within the castrate range for the majority of patients.

Studies IV and V

LUPRON DEPOT 22.5 mg (3-Month SR) was found to be effective in suppressing serum testosterone and maintaining it at the castrate level.

Serum testosterone

Following the initial depot injection, the characteristic increase in mean testosterone over the pretreatment level occurred on Day 4, followed by a steady decline to the castrate range by Week 3. The median time to onset of castrate levels was 22 days. Testosterone suppression was sustained throughout each 12-week dosing interval. After falling into the castrate range, mean testosterone remained well within the castrate range throughout the 12-week interval.

As expected elevated pretreatment levels of alkaline phosphatase (AP) and prostatic specific antigen (PSA) reflected the presence of bony metastatic disease and the general prostatic cancer status respectively. Decreases and/or normalization (in AP and PSA) during treatment reflected the continuing presence of, and presumably the treatment related reduction in bony metastatic disease and/or improvement in the general prostatic cancer status.

Objective response

According to the tumour response rating of the patients, an 85% "no progression" rate (based on best objective response) was achieved during the 24-week treatment period. Complete response was achieved in 1% of the patients, 37% patients had a partial response and 47% patients showed a stable condition.

Eighty (85%) patients responded favourably to the treatment.

Of the 83% of the patients who completed the first 24 weeks of treatment, and continued with the long-term phase of the study, only 17% of the patients discontinued from the study prior to receiving the third injection. Four (4%) patients received only the first injection, 12 (13%) patients received only two injections and 78 (83%) patients received the third injection. Only six patients prematurely terminated the treatment at least in part due to an adverse event. Among those, adverse event was not the primary reason to stop the treatment in four patients. Only one patient discontinued the treatment due to intolerable hot flashes which was, according to the investigator, treatment related.

Laboratory values

LUPRON DEPOT (3-Month SR) has not clinically affected the mean systolic or diastolic blood pressure. Nor the effect on the mean pulse rate is indicative of a clinically significant trend. However, mean body weight significantly increased (p < 0.001) during the treatment. These results were not unexpected, since patients generally showed clinical improvement with treatment during the study. The effect on clinical laboratory determinations [hemogram, white blood cell (WBC), % basophils, total-, high-density lipoprotein (HDL)-, low-density lipoprotein (LDL)-cholesterol, triglycerides, serum glutamate pyruvate transaminase (SGPT), phosphorus, sodium and glucose) were often attributed, by the investigator, to the underlying disease state, to non-fasting blood collection, or as being consistent with the age and status of the patient population studied. As expected, pretreatment levels of alkaline phosphatase reflected the presence of bony metastatic disease. Changes during treatment reflected the continuing presence of, and presumably the treatment-related reduction, in bony metastatic disease.

Safety

Ninety (96%) patients reported adverse events. The most common adverse event was vasodilatation or hot flashes, occurring in 59% of the patients. Among the 94 evaluable patients, only 25% patients classified the adverse event as severe. The overall incidence of severe events (excluding those judged by the investigator as definitely not treatment-related) was low (8 patients, 9%).

The increase in serum testosterone at the beginning of the treatment which has been seen with both the daily injection and the monthly depot formulation, may theoretically result in a transient exacerbation of disease-related symptoms, especially bone pain. Forty-six (49%) of the patients experienced one or more adverse events during the initial two weeks of treatment. Hot flashes was again the most frequently (13%) reported event during this time. Seven (7%) patients reported severe events during this time.

In summary, the leuprolide acetate depot injection releases leuprolide acetate at an apparently steady state; its efficacy in the treatment of advanced prostatic cancer does not differ from the efficacy of the daily subcutaneous injection.

Study VI

In an open-label, non-comparative, multicenter clinical study using LUPRON DEPOT 30.0 mg (4-Month SR), 49 patients with stage D2 prostatic adenocarcinoma (with no prior treatment) were enrolled. The study design and patient demographics is shown in Table 2. The objectives were to determine whether a 30.0 mg depot formulation of leuprolide acetate injected once every 16 weeks would reduce and maintain serum testosterone levels at castrate levels (≤ 50 ng/dL), and to assess the safety of the formulation. The study was divided into an initial 32-week treatment phase and a long-term treatment phase. Serum testosterone levels were determined biweekly or weekly during the first 32 weeks of treatment. Once the patient completed the initial 32-week treatment period, treatment continued at the investigator’s discretion with serum testosterone levels being done every four months prior to the injection.

In the majority of patients, testosterone levels increased 50% or more above the baseline during the first week of treatment. Mean serum testosterone subsequently suppressed to castrate levels within 30 days of the first injection in 94% of patients and within 43 days in all 49 patients during the initial 32-week treatment period. The median dosing interval between injections was 112 days. One escape from suppression (two consecutive testosterone values > 50 ng/dL after castrate levels achieved) was noted at Week 16. In this patient, serum testosterone transiently increased to above the castrate range following the second depot injection (Week 16) but returned to the castrate level by Week 18. No adverse events were associated with this rise in serum testosterone. A second patient had a rise in testosterone at Week 17, then returned to the castrate level by Week 18 and remained there through Week 32. In the long-term treatment phase, two patients experienced testosterone elevations, both at Week 48. Testosterone for one patient returned to the castrate range at Week 52, and one patient discontinued the study at Week 48 due to disease progression.

Secondary efficacy endpoints evaluated in the study were the objective tumor response as assessed by clinical evaluations of tumor burden (complete response, partial response, objectively stable and progression) and elevations of changes in prostatic involvement and PSA. These evaluations were performed at Weeks 16 and 32 of the treatment phase. The long-term treatment phase monitored PSA at each visit (every 16 weeks). The objective tumor response analysis showed “no progression” (i.e. complete or partial response, or stable disease) in 86% (37/43) of patients at Week 16, and in 77% (37/48) of patients at Week 32. Local disease improved or remained stable in all patients evaluated at week 16 and/or 32. For patients with elevated baseline PSA, 50% (23/46) had a normal PSA (< 4.0 ng/mL) at Week 16, and 51% (19/37) had a normal PSA at Week 32.

Using historical comparisons, the safety and efficacy of LUPRON DEPOT 30.0 mg (4-Month SR), appear similar to the other depot formulations.

Detailed pharmacology

Leuprolide acetate is an analog of gonadotropin-releasing hormone (Gn-RH). It was found to have antireproductive properties on chronic administration at high doses, interfering with gonadal steroidogenesis. It produces a reversible regression of steroid-dependent reproductive tissues in both male and female, in a manner analogous to that produced by gonadectomy or by antiandrogenic and antiestrogenic drugs.

Animal Pharmacology

Pharmacodynamics

LUPRON

Several studies in rats were conducted to determine the effects of prolonged administration of leuprolide.

In two non-tumor studies, leuprolide showed in male rats a marked reduction of LH and FSH, accompanied by decreased plasma testosterone at 20 mcg/twice a day for 106 days in the first study and at 20 and 100 mcg/twice a day for 160 days in the second study.

In a tumor study, in male rats implanted with R3327-G prostatic carcinoma, a daily dose of leuprolide at 1, 50 or 1000 mcg/kg for 20 days showed a significant reduction in the tumor growth rate, and enhanced the survival of the animals.

Leuprolide has also been tested in female rats having mammary tumors induced by the administration of 7-12-dimethylbenz[α]-anthracene (DMBA). Doses of leuprolide used ranged from 0.01 mcg to 10 mcg twice a day, up to 31 days. Except for 0.01 mcg which was a "no- effect-dose", leuprolide produced regression of tumor growth similar to the effects seen in the castrate control.

Pharmacokinetics

LUPRON DEPOT

Pharmacokinetic behaviors of leuprolide acetate for depot suspension were studied in rats and dogs.

In rats, release kinetics after subcutaneous and intramuscular injections, exhibited a pseudo-zero- order kinetics for one month in a dose ranging from 3 to 30 mg/kg; the release rate at a dose of 3 mg/kg was 2.8% of dose/day. Serum levels for leuprolide showed a sharp increase immediately after injection, result of an initial burst of the drug, accompanied by an initial flare up of testosterone level. Serum level for leuprolide and testosterone decreased to below normal level, and were sustained at a suppressed level for over six weeks.

In dogs, serum level profiles showed essentially the same pattern.

In a series of experiments with multiple administration (once every four weeks), serum testosterone levels in rats at a dose of 3 mg/kg and those in dogs at 1.5 mg/kg did not show any flare-up at the second and third injection, and continued to be maintained at the suppressed levels. This study demonstrates that leuprolide acetate for depot suspension releases the drug at a constant rate for one month and has a long acting potency.

In another study, the effects of leuprolide acetate for depot suspension on accessory sex organ weights and hormone levels in adult male rats were compared to those produced by leuprolide acetate solution with subcutaneous administration. One group of rats were given 0.2, 1.0 and 5.0 mg/kg/day leuprolide acetate solution for four weeks; the other group received 0.6, 3.0 and 15 mg/kg leuprolide acetate for depot suspension once a week for four weeks. The reduction of organ weights and hormone levels was found more significant with the depot formulation than with the solution.

In another study with rats, the effects of a single administration of leuprolide acetate for depot suspension at doses of 0.03, 0.3 and 3 mg/kg intramuscular, and 3 mg/kg subcutaneously on genital organ weights, were compared to those of the subcutaneous daily injection of 100 mcg/kg/day of solution for two weeks. Results showed that at the beginning of treatment, there was a slight increase, but over the remaining two-week treatment period, the organ weights decreased in dose-related fashion.

Sustained serum drug level, inhibition of steroidogenesis, drastic suppression of the growth of the reproductive organs were observed over a three month period when LUPRON DEPOT (3-Month SR) formulation was studied in rats and dogs.

Human Pharmacology

Pharmacodynamics

With chronic administration, leuprolide had demonstrated a reduction in gonadotropins and sex steroids.

After an initial transient increase in testosterone or estradiol level, leuprolide produces a marked suppression of these levels as well as an inhibition of mammary and prostate tumor growth, and atrophy of the reproductive organs.

This decrease is maintained at castrate levels, as long as treatment continues.

There was no evidence of a dose-response relationship in the testosterone level with doses of 1 mg or 10 mg/day. See CLINICAL TRIALS.

Pharmacokinetics

The absorption, metabolism, distribution, and excretion of leuprolide acetate in humans have not been fully established. See ACTION AND CLINICAL PHARMACOLOGY.

Absorption
LUPRON

The pharmacokinetic profile of leuprolide has been characterized in a single-dose, randomized, two-period, cross-over bioavailability study after administration of 1 mg doses by subcutaneous and by intravenous route in healthy male volunteers. Mean leuprolide plasma level curves were characteristic for each route. Mean levels during earlier sampling times were generally higher after the intravenous regimen, while levels during the later sampling times were generally higher after the subcutaneous regimen. The absolute bioavailability based on the ratio of the mean area under the curve (AUC) for subcutaneous/intravenous was 0.94 with a range of 0.70 to 1.24.

The mean plasma half-life was 2.9 hours. The study demonstrates that the bioavailability of leuprolide after subcutaneous administration was comparable to that of intravenous administration.

LUPRON DEPOT

The pharmacokinetic profile of LUPRON DEPOT has been characterized in an open, single- dose study in 10 orchiectomized prostatic cancer patients given 7.5 mg (1-Month SR) intramuscularly. Blood plasma levels were measured over an 8-week period.

After an initial burst, mean plasma leuprolide acetate concentrations declined to approximately 0.8 ng/mL within four days after the injection and remained basically stable for 2.5 weeks. Prolonged plasma concentrations were achieved with all but one patient with detectable plasma levels up to 4 weeks. Approximately 85 to 100% of the observed 8-week AUC was obtained for each patient after the first four weeks. After 8 weeks, plasma levels were essentially undetectable in all patients.

An estimate of the absolute bioavailability from this dosage form was approximately 90% when compared to an equivalent intravenous solution dose used in another study.

The pharmacokinetic profile of LUPRON DEPOT 22.5 mg (3-Month SR) was characterized in 23 orchiectomized prostate cancer patients. Following a single injection of the three-month formulation of LUPRON DEPOT 22.5 mg (3-Month SR), a mean peak plasma leuprolide concentration of 48.9 ng/mL was observed at four hours and then declined to 0.67 ng/mL at 12 weeks. Leuprolide appeared to be released at a constant rate following the onset of steady- state level during the third week after dosing, providing steady plasma concentrations through the 12-week dosing interval. Detectable levels of leuprolide were present at all measurement points in all patients during this 12-week period. The initial burst, followed by the rapid decline to a steady-state level, was similar to the release pattern seen with the monthly formulation.

The pharmacokinetic profile of LUPRON DEPOT 30.0 mg (4-Month SR) was characterized in 16 orchiectomized prostate cancer patients. Following a single injection of the four month formulation of LUPRON DEPOT 30.0 mg (4-Month SR), a mean peak plasma leuprolide concentration of 59.3 ng/mL was observed at four hours and the mean concentration then declined to 0.30 ng/mL at 16 weeks.

Leuprolide appeared to be released at a constant rate following the onset of steady-state levels during the fourth week after dosing, providing steady plasma concentrations throughout the 16-week dosing interval. However, intact leuprolide and an inactive major metabolite could not be distinguished by the assay employed in the study. The initial burst, followed by the rapid decline to a steady-state level, was similar to the release pattern seen with the other depot formulations.

Toxicology

Acute Toxicity

LUPRON

Acute studies were conducted in rats and mice at 100 mg/kg/day. Only signs of decreased motor activity, dyspnea, and excessive scratching were reported; the LD50 is greater than 100 mg/kg/day in rats and mice.

LUPRON DEPOT

Mice and rats were given leuprolide acetate for depot suspension with different routes of administration: oral, intraperitoneal and subcutaneous (doses of 5 g/kg) and intramuscular (doses of 2 g/kg). No death occurred. The LD50 was concluded to be greater than 5 g/kg for intraperitoneal and subcutaneous routes and 2 g/kg for the intramuscular route.

Long-Term Toxicity

A series of subchronic and chronic toxicity studies conducted in mice, rats, and monkeys with daily subcutaneous injections of leuprolide acetate resulted in atrophy of the sex organs in both male and female animals. Reduced serum levels of gonadotropin hormones were observed in rats and monkeys following administration of leuprolide for 90 days.

LUPRON

Mouse

Maximum tolerated dose studies (prelude to carcinogenicity studies) were conducted in mice. The mice were dosed subcutaneously with 0, 20, 60, 200 and 600 mg/kg/day. Marked skin irritation at injection sites was observed in mice dosed with 200 and 600 mg/kg/day. Hypertrophy of anterior pituitary cells were observed in female mice dosed with 200 mg/kg/day but not at 600 mg/kg/day. Sex organ atrophy, secondary to the drug pharmacologic effects, were observed in all treated male and female mice. The maximum tolerated dose in mice was 60 mg/kg/day.

Rat

Marked pharmacologic effects consisting of atrophy of primary and secondary sex organs in both sexes were observed in rats dosed with 1 to 4 mg/kg/day of leuprolide for 90 days. No overt toxic effects were observed. The "no-toxic-effect" dosage was 4 mg/kg/day.

Maximum tolerated dose studies (prelude to carcinogenicity studies) were conducted in rats. Rats were dosed subcutaneously with 0, 10, 30, 100 and 300 mg/kg/day for 90 days. Drug related pituitary hyperplasia and hypertrophy, atrophy of sex organs (both sexes) and marked skin irritation at the injection sites were observed in rats. As a result, no maximum tolerated dose was established by the study.

Monkey

Rhesus monkeys dosed subcutaneously with 0, 1, 2 and 4 mg/kg/day for 90 days exhibited marked atrophy of the primary and secondary sex organs of both sexes. The reproductive effects were consistent with the pharmacologic action of the drug. The "no-toxic-effect" dosage was 4 mg/kg/day as no overt toxicity was observed.

Leuprolide acetate was administered subcutaneously to cynomolgus monkeys once daily at dosages of 0, 0.6, 4.0 and 10 mg/kg/day for one year. Atrophy of sex organs of both sexes was the principal finding. These changes were ascribed to the pharmacologic activity of the drug. The "no-toxic-effect" dose was 10 mg/kg/day.

LUPRON DEPOT

Rat

Leuprolide acetate for depot suspension was administered intramuscularly to three groups of male rats at doses from 10, 30 and 100 mg/kg/week (corresponding to 0.8, 2.4 and 8.0 mg/kg/week of leuprolide acetate injection) once a week for 13 weeks. Rats dosed at 100 mg/kg/week showed atrophy of testes; in addition white spots were noted at the injection sites. The atrophy of the testes was reported to be due to the hormonal action of leuprolide acetate injection; the "no-toxic-effect" dose was considered to be 100 mg/kg/week.

In another toxicity study, male rats were given leuprolide acetate for depot suspension subcutaneously once a week for three weeks, at doses of 30 mg/kg/week (corresponding to 2.4 mg/kg/week of leuprolide acetate injection). Atrophy of the testes, and a slight induration were noted. The "no-toxic effect" dose was considered to be 30 mg/kg/week.

In a third study, leuprolide acetate for depot suspension was given subcutaneously to groups of male and female rats, at doses of 0, 10, 30 and 100 mg/kg/week once a week for 13 weeks (corresponding to 0, 0.8, 2.4 and 8 mg/kg/week of leuprolide acetate injection). Atrophy of the testes was noted, with induration at injection site; in female rats, the vagina failed to open throughout the dosing period. Leuprolide acetate for depot suspension produced changes related to the expected pharmacologic effects. The "no-toxic-effect" dose was considered to be 100 mg/kg/week.

Dog

In two different studies, female and male beagle dogs were given leuprolide acetate for depot suspension subcutaneously for 13 weeks, once a week at doses of 10, 30, 100 mg/kg/week, corresponding to 0.8, 2.4 and 8 mg/kg/week leuprolide acetate injection. No death was reported. Signs and symptoms include inflammatory lesions at the injection sites, and atrophic changes of the primary and accessory sex glands. The injection site change, seen in both control and test groups, was induced by the microcapsule, not leuprolide, and was reversible.

Special Studies

LUPRON DEPOT

Rabbit

In a preliminary study, male rabbits were given single injections (1 mL/animal) of a 15% suspension of leuprolide acetate for depot suspension into the subcutaneous tissue of the abdomen to assess local irritation.

Deposition of the test drug at site of injection was noted at 2 and 14 days after the injection, along with slight hemorrhage and dilatation of capillaries at 50 days after the injection. Leuprolide acetate for depot suspension was reported not to produce significant subcutaneous irritation in rabbits in this study.

In a second irritation study, male rabbits were injected once or four successive times with leuprolide acetate for depot suspension (15% suspension) by intramuscular administration. Results were compared to those obtained with placebo-microcapsule or a 0.75% solution of acetic acid as the positive control. Deposition at injection sites, and slight irritation changes (hemorrhage, edema, inflammation) were noted: leuprolide acetate for depot suspension produced the same effects with same the degree as the placebo-microcapsule, but these are less than those of the positive control (0.75% acetic acid), and their severity were not potentiated by four repeated injections.

The injection-site toxicity and irritation effects of LUPRON DEPOT (3-Month SR) were studied in rabbits. The rabbits were administered with intramuscular and subcutaneous injections at doses of 11.25 mg/mL for intramuscular injection and 5.64 mg/mL for subcutaneous injection. Intramuscular injection was in the left vastus lateralis muscle, and subcutaneous injection was in the abdominal region. Only mild irritative changes such as mild hemorrhage and degeneration of the muscle fiber were seen two days after the injection. Moreover, granulation tissue composed of macrophages and multinucleated giant cells was observed. The size of granulation tissue observed was decreased 13 weeks after the injection. Therefore, these changes were characterized mainly by foreign body reactions caused by the persistence of the microcapsule formulation.

Guinea Pig

Two studies were performed to evaluate the potential of leuprolide acetate for depot suspension to produce either systemic anaphylaxis or delayed hypersensitivity reactions in guinea pigs.

Preliminary antigenicity study. Leuprolide acetate for depot suspension was given to guinea pigs at a dose of 123 mg/kg every two weeks by intramuscular route four times, and once by subcutaneous route two weeks after the last intramuscular dose. Results were compared to controls treated with placebo-microcapsule 122 mg/kg intraperitoneally, or with ovalbumin 5 mg/animal intravenously. No systemic anaphylactic reactions were observed with animals treated with leuprolide acetate for depot suspension and placebo-microcapsule, but some induced equivocal weak antibody production was noted.

In a second study, the sensitization potential of leuprolide acetate for depot suspension at doses of 50 mg/animal/dosing by intramuscular (systemic anaphylaxis) or at doses of approximately 7.2 mg/animal/dosing (0.05 mL of a 144.23 mg/mL of suspension) intradermal (delayed hypersensitivity), were compared to those seen with gelatin, egg albumin or captan. No signs of anaphylactic reactions nor delayed hypersensitivity were observed for leuprolide acetate for depot suspension, while signs of anaphylactic reactions (such as nose scratching, sneezing, dyspnea or local irritation) were noted with other compounds.

Mutagenicity and Carcinogenicity

Mutagenicity

LUPRON

Leuprolide has been studied in vitro and in vivo, using bacterial and mammalian systems.

In vitro assays using Salmonella and Saccharomyces with and without the presence of liver microsomal enzyme from Aroclor-1254 induced rats, no signs of mutagenicity have been observed.

Leuprolide was non-mutagenic in vivo cytogenic assay in rats or in the Mouse Dominant Lethal assay at doses of 0, 1, 2 and 4 mg/kg administered subcutaneously.

Both in vitro and in vivo studies have provided no evidence of a mutagenic potential of leuprolide.

LUPRON DEPOT

In the Ames Test, using S. typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, and E. coli strain WP2hcr, leuprolide acetate for depot suspension was found not mutagenic at dosing ranging from 0.03 to 10 mg/plate, irrespective of treatment with mammalian metabolic activation system (S-9 mix).

Carcinogenicity

LUPRON DEPOT

Two rodent carcinogenicity studies were conducted for two years with daily doses of 0.6, 1.5, and 4 mg/kg/day in the rat, and with 0.6, 6, and 60 mg/kg/day in the mouse.

In rats, a dose-related incidence of pituitary hyperplasia, hypertrophy and benign pituitary adenomas were noted at 12 month necropsy, while a statistically significant dose-related incidence of benign pituitary adenomas was observed in both male and female rats after 24 months when the drug was administered subcutaneously at high daily doses (0.6 to 4 mg/kg).

In mice, no drug-induced neoplastic changes or pituitary abnormalities were observed at doses as high as 60 mg/kg for two years.

Patients have been treated with leuprolide for up to three years with doses as high as 10 mg/day, and for two years with doses as high as 20 mg/day. Clinical signs of pituitary abnormalities have not been observed in any of these patients.

Reproduction and Teratology

Fertility and Reproduction

LUPRON DEPOT

Fertility and reproductive performance studies cannot be conducted with leuprolide, because the compound affects the pituitary-gonadal axis and influences endocrine reproductive organs. As a result, there would be a decrease in fertility and reproduction.

Clinical and pharmacologic studies with leuprolide acetate and similar analogs have shown full reversibility of fertility suppression when the drug is discontinued after continuous administration for periods of up to 24 weeks.

Although no clinical studies have been completed in children to assess the full reversibility of fertility suppression, animal studies (prepubertal and adult rats and monkeys) with leuprolide acetate and other GnRH analogs have shown functional recovery. However, following a study with leuprolide acetate, immature male rats demonstrated tubular degeneration in the testes even after a recovery period. In spite of the failure to recover histologically, the treated males proved to be as fertile as the controls. Also, no histologic changes were observed in the female rats following the same protocol. In both sexes, the offspring of the treated animals appeared normal. The effect of the treatment of the parents on the reproductive performance of the F1 generation was not tested. The clinical significance of these findings is unknown.

Teratology

LUPRON DEPOT

Leuprolide administered to pregnant rats at dosages of 0, 1, 3 and 10 mcg/kg/day from Gestational Day 6 to Gestational Day 15 (major period of organogenesis) was not teratogenic. At 10 mcg/kg/day, leuprolide increased the incidence of resorptions; surviving fetuses showed no abnormalities. The "no-toxic-effect" dosage was 3 mcg/kg/day.

Leuprolide increased the incidence of embryonic resorptions in pregnant rabbits when dosed with 0, 0.1, 0.3 or 1.0 mcg/kg/day during the period of major organogenesis, i.e., Gestational Day 6 through Gestational Day 18. Surviving fetuses showed no abnormalities.