Draft Guidance on Doxorubicin Hydrochloride

Contains Nonbinding Recommendations

Draft Guidance on Doxorubicin Hydrochloride

This draft guidance, once finalized, will represent the Food and Drug Administration's (FDA's) current

thinking on this topic. It does not create or confer any rights for or on any person and does not operate

to bind the FDA or the public. You can use an alternative approach if the approach satisfies the

requirements of the applicable statutes and regulations. If you want to discuss an alternative approach,

contact the Office of Generic Drugs.

Active Ingredient: Doxorubicin hydrochloride

Dosage Form; Route: Liposome injection; intravenous

Recommended Studies: Two studies

When the test and reference pegylated liposome products

• have the same drug product composition and

• are manufactured by an active liposome loading process with an ammonium sulfate

gradient and

• have equivalent liposome characteristics including liposome composition, state of

encapsulated drug, internal environment of liposome, liposome size distribution, number

of lamellar, grafted PEG at the liposome surface, electrical surface potential or charge,

and in vitro leakage rates.

The following clinical and in vitro studies are recommended to demonstrate bioequivalence:

In Vivo Bioequivalence Study:

1. Type of study: Fasting*

Design: Single-dose, two-way crossover in vivo

Strength: 50 mg/vial or 20 mg/vial

Dose: 50 mg/m2

Subjects: Ovarian cancer patients whose disease has progressed or recurred after

platinum-based chemotherapy and who are already receiving or scheduled to start therapy

with the reference listed drug (RLD) or the reference standard product.

Additional comments:

• Doxorubicin is a cytotoxic drug. Therefore, a Bio-IND is required for

bioequivalence studies of a doxorubicin liposome injection to ensure the safety of

human test subjects.

• The two arms of the crossover study are to be conducted on two of the days when

the patients are scheduled to receive their usual therapy so that the treatment

regimen is not altered or delayed.

• The standard of care treatment regimen should not be altered except to randomize

the patients to the test or reference therapy on the specified dosing days.

• Given that the dosage is every 4 weeks, two consecutive treatment cycles should

be used for the two treatment periods.

Recommended Feb 2010; Revised Nov 2013, Dec 2014

• Any concomitant medications must be exactly the same in both periods of the

study.

• Due to concerns about cardiac toxicity, cardiac status should be documented at

baseline.

• Any patient whose weight changes during the study requiring a ±5% dose

adjustment must be discontinued from the study and excluded from the analysis.

Exclusion Criteria:

• Prior doxorubicin exposure that would result in a total lifetime exposure of 550

mg/m2

or more after four cycles of treatment.

• The protocol must exclude patients with significantly impaired hepatic function in

their exclusion criteria.

• Patients who have a history of hypersensitivity reactions to a conventional

formulation of doxorubicin HCl or the components of the RLD or reference

standard should not be entered into the study.

• Females should not be pregnant or lactating.

• Patient is < 18 years of age or > 75 years of age.

• Active opportunistic infection with mycobacteria, cytomegalovirus, toxoplasma,

P. carinii or other microorganism if under treatment with myelotoxic drugs.

• Clinically significant cardiac, liver or kidney disease.

* If the health conditions of patients prevent fasting, the sponsor can provide a non-high-fat diet during the proposed study. Alternatively, the treatment can be initiated 2 hours

after a standard (non-high-fat) breakfast.

Analytes to measure (in appropriate biological fluid): Free doxorubicin and liposome

encapsulated doxorubicin.

Bioequivalence based on (90% CI): AUC and Cmax for free doxorubicin and liposome

encapsulated doxorubicin.

Note: the pivotal bioequivalence study should be conducted using test product produced

by the proposed commercial scale manufacturing process.

In Vitro Study:

2. Type of study: Liposome Size Distribution

Design: in vitro bioequivalence study on at least three lots of both test and reference

products

Parameters to measure: D10, D50, D90

Bioequivalence based on (95% upper confidence bound): D50 and SPAN [(i.e. D90-

D10)/D50] or polydispersity index using the population bioequivalence approach.

Recommended Feb 2010; Revised Nov 2013, Dec 2014 2

Dissolution test method and sampling times: The dissolution information for this drug product

can be found on the FDA-Recommended Dissolution Methods website available to the public at

the following location: /scripts/cder/dissolution/. Conduct

comparative dissolution testing on 12 dosage units each of all strengths of the test and reference

products. Specifications will be determined upon review of the abbreviated new drug application

(ANDA).

Additional information:

Same drug product composition

Being a parenteral drug product, a generic doxorubicin HCl liposome injection must be

qualitatively and quantitatively the same as the RLD or reference standard, except differences in

buffers, preservatives and antioxidants provided that the applicant identifies and characterizes

these differences and demonstrates that the differences do not impact the safety/efficacy profile

of the drug product. Currently, FDA has no recommendations for the type of studies that would

be needed to demonstrate that differences in buffers, preservatives and antioxidants do not

impact the safety/efficacy profile of the drug product.

Lipid excipients are critical in the liposome formulation. ANDA sponsors should obtain lipids

from the same category of synthesis route (natural or synthetic) as found in the RLD or reference

standard. Information concerning the chemistry, manufacturing and control of the lipid

components should be provided at the same level of detail expected for a drug substance as

suggested in the liposome drug products draft guidance1. ANDA sponsors should have

specification on lipid excipients that are similar to those used to produce the RLD or reference

standard. Additional comparative characterization (beyond meeting specifications) of lipid

excipients including the distribution of the molecular species should be provided.

Active liposome loading process with an ammonium sulfate gradient

In order to meet the compositional equivalence and other equivalence tests, an ANDA sponsor

would be expected to use an active loading process with an ammonium sulfate gradient. The

major steps include 1) formation of liposomes containing ammonium sulfate, 2) liposome size

reduction, 3) creation of ammonium sulfate gradient, and 4) active drug loading. An active

loading process uses an ammonium sulfate concentration gradient between the liposome interior

and the exterior environment to drive the diffusion of doxorubicin into the liposomes2,3.

Sponsors should use a Quality by Design approach to identify critical material attributes and

critical process parameters, and guide process optimization. It is recommended to identify the

1 Draft guidance for industry: Liposome drug products chemistry, manufacturing, and controls; human

pharmacokinetics and bioavailability; and labeling documentation, FDA (2002),

/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/

2 A. Gabizon, H. Sheemda, Y. Barenholz. Pharmacokinetics of pegylated liposome doxorubicin: review of animal

and human studies. Clin Pharmcokinet 42(5): 419-436 (2003)

3 F. Martin. Product evolution and influence of formulation on pharmaceutical properties and pharmacology,

Advisory Committee for Pharmaceutical Science Presentation (Jul 2001),

/ohrms/dockets/AC/01/slides/3763s2_08_.

Recommended Feb 2010; Revised Nov 2013, Dec 2014 3

critical process parameters and critical material attributes by evaluating the sensitivity of

liposome characteristics to changes in process parameters and attributes. The optimal values of

critical process parameters should be selected based on comparison of resulting liposome

characteristics to those of the RLD or reference standard.

Equivalent liposome characteristics

As with other locally acting products with complex bioequivalence requirements (such as nasal

sprays and inhalation products), in vitro liposome characterization should be conducted on at

least three batches of the ANDA and the RLD or reference standard products (at least one

ANDA batch should be produced by the commercial scale process and used in the in vivo

bioequivalence study). Attributes that should be included in the characterization of ANDAs

claiming equivalence to the RLD or reference standard are:

• Liposome composition: Liposome composition including lipid content, free and encapsulated

drug, internal and total sulfate and ammonium concentration, histidine concentration, and

sucrose concentration should be measured. The drug-to-lipid ratio and the percentage of drug

encapsulation can be calculated from liposome composition values.

• State of encapsulated drug: The doxorubicin in the RLD or reference standard is largely in

the form of a doxorubicin sulfate precipitate inside the liposome. The generic doxorubicin

HCl liposome must contain an equivalent doxorubicin precipitate inside the liposome.

• Internal environment (volume, pH, sulfate and ammonium ion concentration): The internal

environment of the liposome, including its volume, pH, sulfate and ammonium

concentration, maintains the precipitated doxorubicin. The measurements of total and free

concentrations of components (including sulfate ions) described in liposome composition

section allow the inference of the internal concentration inside the liposome.

• Liposome morphology and number of lamellae: Liposome morphology and lamellarity

should be determined as drug loading, drug retention, and the rate of drug release from the

liposomes are likely influenced by the degree of lamellarity.

• Lipid bilayer phase transitions: Equivalence in lipid bilayer phase transitions will contribute

to demonstrating equivalence in bilayer fluidity and uniformity. The phase transition profiles

of the raw lipid excipients and liposomes should be comparable to those of the RLD or

reference standard.

• Liposome size distribution: Liposome size distribution is critical to ensuring equivalent

passive targeting. The ANDA sponsor should select the most appropriate particle size

analysis method to determine the particle size distributions of both test and reference product.

The number of liposome product vials to be studied should not be fewer than 30 for each of

the test and reference products (i.e., no fewer than 10 from each of three batches). See

recommended study 2 (above) for details of the recommended statistical equivalence tests.

Recommended Feb 2010; Revised Nov 2013, Dec 2014 4

• Grafted PEG at the liposome surface: The surface-bound methoxypolyethylene glycol

(MPEG) polymer coating protects liposomes from clearance by the mononuclear phagocyte

system (MPS) and increases blood circulation time. The PEG layer thickness is known to be

thermodynamically limited and estimated to be in the order of several nanometers. The PEG

layer thickness should be determined.

• Electrical surface potential or charge: Surface charge on liposomes can affect the clearance,

tissue distribution, and cellular uptake. Liposome surface charge should be measured.

• In vitro leakage under multiple conditions: In vitro drug leakage testing to characterize the

physical state of the lipid bilayer and encapsulated doxorubicin should be investigated to

support a lack of uncontrolled leakage under a range of physiological conditions and

equivalent drug delivery to the tumor cells. Below are some examples of proposed

conditions.

Table 1. Examples of in vitro leakage conditions of doxorubicin liposomes

In Vitro Drug Purpose Rationale

Leakage Condition

At 37ºC in 50% Evaluate liposome Plasma mostly mimics blood conditions.

human plasma for stability in blood

24 hours circulation.

At 37ºC with pH Mimic drug release in Normal tissues: pH 7.3

values 5.5, 6.5, and normal tissues, around Cancer tissues: pH 6.6

7.5 for 24 hours in cancer cells, or inside Insider cancer cells (endosomes and

buffer cancer cells lysosomes): pH 5-6 (Endosome and lysosomes

of cancer cells may be involved in liposome

uptake and induce drug release).

At a range of Evaluate the lipid The phase transition temperature (Tm) of lipids

temperatures (43ºC, bilayer integrity is determined by lipid bilayer properties such

47ºC, 52ºC, 57ºC) in as rigidity, stiffness and chemical composition.

pH 6.5 buffer for up Differences in release as a function of

to 12 hours or until temperature (below or above Tm) will reflect

complete release small differences in lipid properties

At 37ºC under low-Evaluate the state of Low-frequency ultrasound (20 kHz) disrupts

frequency (20 kHz) encapsulated drug in the lipid bilayer via a transient introduction of

ultrasound for 2 the liposome. pore-like defects and will render the release of

hours or until doxorubicin controlled by the dissolution of

complete release. the gel inside the liposome.

Equivalent in vivo plasma pharmacokinetics of free and encapsulated drug

A Bio-IND is required to conduct bioequivalence studies of doxorubicin liposome injection in

humans since doxorubicin is a cytotoxic drug. We recommend single dose fasting two-way

crossover bioequivalence studies in ovarian cancer patients at 50 mg/m2 dose. Sponsors should

measure both liposome-encapsulated and free doxorubicin to demonstrate the same in vivo

Recommended Feb 2010; Revised Nov 2013, Dec 2014 5

stability of generic liposome formulation and the RLD or reference standard. The studies may be

conducted under either fasted or standard diet conditions depending on patient needs. See

recommended study 1 (above) for details of the recommended statistical equivalence tests.

Recommended Feb 2010; Revised Nov 2013, Dec 2014 6