Lasers in Surgery and Medicine 41:714–720 (2009)

Injectable Treatments for Adipose Tissue: Terminology, Mechanism, and Tissue Interaction Adam M. Rotunda, MD* Division of Dermatology, David Geffen School of Medicine, University of California, Los Angeles, California

Background: Just as injectable fillers have addressed the need for non-surgical methods to restore desired volume, a number of injectable therapies purpor to play a comparable role to reduce undesired volume. Objective: To review published literature on the history, mechanism of action, and tissue interaction of injectable methods that aim to reduce localized collections of fat. Results: Mesotherapy is an injection technique that has medical and cosmetic applications and is often confused with injectable fat loss therapies; injection lipolysis describes non-ablative fat reduction with agents (such as b-agonists) that activate adipocyte lipolytic pathways; and adipolytic therapy using biologic detergents (such as deoxycholate) leads to permanent adipocyte ablation. None of these therapies have been cleared for use in fat reduction by any regulatory authority worldwide. Conclusions: The mechanism of action and tissue effects of injectable fat reducing compounds are diverse but are becoming increasingly understood. Lasers Surg. Med. 41:714–720, 2009. ß 2009 Wiley-Liss, Inc. Key words: deoxycholate; phosphatidylcholine; adipolysis; injection lipolysis; mesotherapy; lipodissolve

INTRODUCTION Subcutaneous injections that reduce modest collections of adipose tissue have been most commonly referred to as injection lipolysis and mesotherapy [1–5]. The popularity of colloquially termed ‘‘fat melting’’ injections can be attributed in part to a successful Lipodissolve1 marketing campaign by Fig. (formerly American LipoDissolve, St Louis, MO) commercial treatment centers and the American Society for Aesthetic Lipodissolve (ASAL). As of this writing, however, no commercially available formulations are approved anywhere worldwide for injectable fat removal. The United States Food and Drug Administration (FDA) considers injectable fat reduction ‘‘unapproved drugs for unapproved uses. . .,’’ [6] yet interest persists. Notorious reports of cutaneous infections and necrosis, bankruptcies, medical society warning statements, as well as state legislation banning the procedure [7–14], have contributed to the controversy. Despite the alarm, validation of efficacy and an acceptable safety profile by recent investigations point toward the prospect that a fat reducing injection may one day become available. ß 2009 Wiley-Liss, Inc.

CLARIFICATION OF TERMS AND HISTORY Table 1 attempts to clarify terminology frequently used in discussions and literature relating to injectable methods to reduce fat. Injection Lipolysis Versus Adipolytic Therapy Lipolysis describes the hydrolysis, or degradation, of lipids into their constituent fatty acid and glycerol building blocks [15]. This process occurs within adipocytes or within the vascular space of muscle and fat tissue, and is governed by hormone sensitive lipase (HSL) and lipoprotein lipase (LPL) [15]. HSL is expressed in adipose tissue and is activated by cortisone and catecholamines, which are lipolytic, and inhibited by insulin, which is lipogenic. LPL is located in endothelial walls of capillaries and is responsible for cholymicron (from dietary lipids) and very low-density lipoprotein breakdown. Lipolysis may also be induced by medications and hormones that bind to specific adrenergic receptors (a or b) located on adipocyte membranes [15–18]. The term injection lipolysis suggests injectable methods which ‘‘activate’’ adipocytes to mobilize their fatty acid stores without affecting the integrity of their cell membranes. The focus of this article and indeed the preponderance of literature on the subject of injectable treatments for fat relate to agents that completely ablate or destroy fat by breaking down or solubilizing the fat cell membrane. The most well studied component of these treatments is sodium deoxycholate (DC), a biologic detergent (Fig. 1). Unless noted elsewhere, this compound is present in all formulations that include phosphatidylcholine (PC). It is therefore proposed that the terms injection adipolysis or adipolytic therapy [19] most accurately describe injectable methods that employ detergents to reduce fat. Mesotherapy The term mesotherapy is distinct from adipolytic therapy [2,4,5,21]. Mesotherapy (from the Greek mesos, ‘‘middle’’ and therapeia, ‘‘to treat medically’’) was first introduced in 1952 by French physician Michel Pistor [22] and describes cutaneous injections of minute doses of medication. Meso*Correspondence to: Dr. Adam M. Rotunda, MD, Assistant Clinical Professor, 1401 Avocado Avenue, Suite 810, Newport Beach, CA 92660. E-mail: [email protected] Accepted 23 July 2009 Published online 15 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lsm.20807

Bile salt detergent, DC; PC historically added to DC [2,5]; injections of PC will also contain DC as its solvent, whether or not noted [27–31,33-36,40] First reported by Patricia Rittes, M.D., using Lipostabil1 [21]; DC first used in isolation from PC to reduce lipomas [43] Adipolysisa, adipocytolysis [6,41], adipocyte lysis [2], adipolytic therapy [19], PC/DC or DC Lipodissolve1 Subcutaneous injections of compounds that ablate tissue and solubilize cell membranes

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Fig. 1. Chemical structure of sodium deoxycholate.

PC, phosphatidylcholine; DC, deoxycholate. The FDA has not cleared any pharmaceutical medication for any of the above techniques. a Personal communications, Steven Weiss, M.D., 2006.

b-agonists (isoproterenol), a2 antagonists (yohimbine), phosphodiesterase inhibitors (aminophylline) Derived from lipolysis, a term used in physiology [15] that describes hydrolysis or breakdown of triglyceride in the medical literature Injection lipolysis Subcutaneous injections of compounds that mobilize and reduce fat without lysing or ablating cells

Primarily used in pain management and sports medicine; most recent investigations on melasma and photoaging [5,17,20,23–26]; associated with cutaneous complications [5,7,8,13] Promising but clinical data conflicting and limited [17,18,20]; fat cell remains viable and intact, therefore volume reduction may be transient; the term ‘‘injection lipolysis’’ previously but (technically) incorrectly used to describe DC or PC/DC injections DC solubilizes cell membranes, induces tissue necrosis, inflammation, and permanent volume reduction [2,45,46,51]; PC possesses neither adipolytic nor lipolytic activity [2,16] Anesthetics, herbs, vitamins, vasodilators, NSAIDS, non-cross-linked hylauronic acid, mineral chelators, and other compounds [1,5] Described by Dr. Michel Pistor in France in 1952 [22] Mesotherapy, intradermotherapy A cutaneous injection technique using numerous compounds to locally treat medical and cosmetic conditions [1,5]

Definition/process

TABLE 1. Clarification of Terminology

Terms

History

Examples of medications used

Comments

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therapy has been recognized and traditionally practiced in Europe as a localized treatment in pain medicine, sports medicine, and rheumatology, but has been applied over time to cosmetic medicine for conditions like alopecia, cellulite, photoaging, and scarring [1,4,5,22]. The nature of the target condition determines at what depth (epidermal, dermal, or subcutaneous) the injections (often hundreds) of extemporaneously mixed combinations of vasodilators, anti-inflammatory agents, herbs, hormones, antibiotics, enzymes, or co-enzymes are placed with a syringe, injection gun, or multi-needle device [1,4,5]. Several small, yet well-designed studies have generated renewed interest in this traditional form of mesotherapy as a treatment for a number of aesthetic and medical conditions [23 –26]. Lipostabil1 and Lipodissolve1 Injectable fat reducing techniques emerged in the world literature when Patricia Rittes [27], a dermatologist in Sa˜ o Paolo, Brazil, reported reduction of infraorbital fat using direct, transcutaneous injection with Lipostabil1 (Fig. 2). Lipostabil1 is manufactured by Sanofi-Aventis (Paris, France) and marketed for intravenous use in Europe, South American, and South Africa as an treatment for numerous fat-related disorders (i.e., hyperlipidemia, angina pectoris, diabetic angiopathy). Lipostabil1 consists of soy-derived PC (5%), its solvent sodium deoxycholate (2.5%), dl-a-tocopherol (vitamin E), sodium hydroxide, ethanol, and benzyl alcohol, in sterile water. Additional investigations by Dr. Rittes [28–30] and others [2,21,31–45] have described using Lipostabil1, compounded PC/DC, and compounded DC only for fat reduction in the hips, abdomen, back rolls (known affectionately as ‘‘love handles’’ (Fig. 3) in men or ‘‘bra strap fat’’ in women), dorsocervical region (‘‘buffalo hump’’), neck, jowls, and lipomas (Fig. 4). Two recently conducted prospective, randomized, double-blind clinical trials using PC/DC and DC alone in submental fat [3] (Fig. 5) and hips [41] and have substantiated the safety profile and efficacy reported in case series and retrospective studies. The indications, technique, safety profile, and standards of practice of adipolytic therapy have been recently reviewed [2,3,30]. In the United States, Lipostabil1 is not available and there are no pharmaceutical grade injectable PC/DC or DC medications marketed for any medical or cosmetic

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Fig. 2. A 55-year-old patient with prominent infraorbital fat pads treated with Lipostabil. (A) Before and (B) after four injections sessions. Reproduced from Rittes [27]. Used with permission from Blackwell Publishing. Fig. 4. Ultrasound, with measurements, of a left shoulder lipoma (A) before and (B) 4 months after two injection sessions with DC (1%). Photos courtesy of the author.

condition. Therefore, importing Lipostabil1 is illegal, and there are no approved medications for which physicians can use ‘‘off-label.’’ In response to the growing interest, however, compounding pharmacies have produced formulations similar to Lipostabil1. Caffeine or collagenase or other lipolytic agents may be added to purportedly [18] augment fat reduction. The compounded formulations generally consists of 5% PC combined with 4.2–4.75% DC [2,3,5] or 1–5% DC alone [2,3,32,37,41,43]. In the United States, these compounded medications are

Fig. 3. Fifty-four-year-old patient (A) before and (B) after two injection session with a PC/DC solution into the inferior back roll (‘‘love handles’’). Obtained with permission from Diane Duncan, M.D.

Fig. 5. Patient profile (A) before and (B) 2 months after 5 monthly injection sessions with 1.0 mL of DC (1%) into the submental fat. Photos courtesy of the author.

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legally available [2,5]. The regulations pertaining to compounding vary by state and should be understood before a physician practices the technique [2,5]. Physicians should determine the extent of their malpractice coverage relating to adipolytic injections before treating patients. MECHANISM AND TISSUE INTERACTION Isolation of the Adipolytic Ingredient Early publications [27,42,47] hypothesized that the lecithin-derived phospholipid, PC, was the active fatreducing ingredient in Lipostabil1. This theory was based upon the premise that the mechanism responsible for reducing serum lipids by PC in Lipostabil1 was working in a similar manner to reduce subcutaneous fat tissue. It was further conjectured [16,47] that PC induced a cascade of intracellular signals that led to apoptosis, or that it directly lysed fat cell membranes, emulsified triglycerides, upregulated LPL, and facilitated transit of triglycerides across cell membranes. Yet, none of these theories could be supported experimentally. An unforeseen discovery [45] revealed that DC alone (without PC) produced cell death and cell lysis in vitro (keratinocytes) and ex vivo (pig adipose tissue) equal to that produced by the PC/DC combination (Fig. 6). These data called into question the role of PC as the fat reducing agent by demonstrating that the bile salt, DC, produces nonspecific cell lysis independent of PC. These experimental data have since been corroborated. Gupta et al. [46] demonstrated that DC alone and PC/DC are comparably cytotoxic on cultured adipocytes, endothelial cells, fibroblasts, and skeletal muscle cells (Fig. 7). These data establish that these compounds act in a nonspecific manner on multiple cell types. Schuller-Petrovic et al. [32] translated these laboratory findings into living tissue by performing cell lysis and cell viability studies in vivo (rat adipose tissue) using PC/DC and isolated DC (Fig. 8). The clinical relevance of this experimental work

Fig. 6. MTS cell viability assay measuring living keratinocytes exposed to phosphatidylcholine/deoxycholate (PC/DC) and deoxycholate (DC). Absorbance (OD) is directly related to cell viability. Increasing concentration of either PC/DC or DC alone produces cell death. DC alone profoundly reduces cell viability, with PC producing minimal effect. Modified with permission from Blackwell Publishing.

Fig. 7. Cytototoxic effect of (A) PC/DC and (B) DC alone on adipocytes assessed with MTT colorimetric assay. The lytic effects of DC on keratinocytes seen in Figure 6 can be similarly produced in adipocytes. Cells were treated for 24, 48, and 72 hours. Data points represent the means and standard deviations for comparison with untreated control cells using increasing concentration of reagents. Reproduced from Gupta [46]. Obtained with permission from Springer.

has been validated through studies that demonstrate comparable effects when either DC or PC/DC are injected into lipomas [38,43,44], abdominal [2,37], submental [3], and hip fat [2,3,37–39,41]. Deoxycholic acid is a secondary bile acid produced by intestinal bacteria after the release of primary bile acids (i.e., cholic acid) in the liver [5]. Biologically compatible detergents like DC have been conventionally used to improve the solubility of the major constituents of intravenous medications, such as Amphotericin B (Amphocin1 Pfizer) [5]. Sodium deoxycholate is the solvent for PC in Lipostabil1, as phospholipids like PC are very poorly water soluble [50]. Ionic detergents like DC disrupt the integrity of biological membrane by introducing their polar hydroxyl groups into the cell membrane’s phospholipid bilayer hydrophobic core [51]. In a sequential manner, the process involves first an ‘‘attack’’ of the detergent on the membrane; solubilization of membrane associated proteins; saturation of the membrane with detergent; and finally, with increas-

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Fig. 9. (A) Histological findings of an excised lipoma 48 hours after subcutaneous infiltration with DC (1%), revealing a welldemarcated area of acute inflammation, extravasated erythrocytes, and necrosis adjacent to relatively unaffected adipose tissue (hematoxylin and eosin, original magnification 10
). Courtesy of the author. (B) Inflammation and fibrosis 6 months after abdominal fat injected with 1% DC. Reproduced from Yagina [37]. Used with permission from Blackwell Publishing. Fig. 8. Effects of DC (2.5%) on (A) rat fat cell membrane integrity and (B) cell viability after repetitive dosing. The effects were observed after 30 days following application of DC on days 0, 7, and 28. Triton (TTX) 0.5% served as positive control. These data translate the experimental data summarized in Figures 6 and 7 into a living model. Reproduced from Schuller-Petrovic [32]. Obtained with permission from Medical Insight, Inc.

ing detergent concentration, membrane breakdown, and solubilysis [51–54]. Detergents and Tissue Interactions—Implications on Efficacy and Safety As it is well recognized in sclerotherapy, detergent effects on tissue are profound [55]. Fundamentally akin to this, subcutaneously injected DC necroses tissue as a result of its cytotoxic, detergent effects on the cellular membranes. Almost immediately, inflammation and edema manifest clinically. Living and ex vivo animal human tissue exposed to DC, as well as PC/DC combinations demonstrate fat cell lysis; fat, muscle, and collagen necrosis; erythrocytes extravasation; a mixed infiltrate consisting of polymorphonuclear leukocytes, lymphocytes,

macrophages, and multinucleated giant cells; and fibrosis (Fig. 9) [2,5,29,32,37–39,43,45,48]. Controlled fat ablation and moderate degrees of post-inflammatory scarring (fibrosis) are desired for volume reduction and tissue tightening, respectfully [2,3,21]. Prior clinical and experimental data [2,45,46] demonstrate that ablative effects of DC or PC/DC solutions are non-specific, suggesting that unintentional administration into non-adipose tissue (i.e., skin, muscle, nerve) could lead to harmful outcomes. However, a recent rat study did not reveal any gross, histological, or functional evidence of damage in tibial nerves injected with Lipostabil [56]. Although limited in number, two controlled clinical studies reported transient injection site erythema, tenderness, and edema as the primary adverse reactions [3,41]. Other published reports (primarily open-label series) substantiate that adipolytic therapy is generally well tolerated [2,3,5,27–44]. There does appear to be, however, a dosedependent response to DC, whereby increasing DC concentrations produces increasing degrees of cytotoxicity [45,46], and subcutaneous fat necrosis [2,37,43]. These data collectively suggest some safe therapeutic margin, where

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maximum benefit is achieved with minimal risk, which should be defined and exploited. Factors such as injection technique [2,30,47,49], dose (volume and concentration) [2,3,30,45,47,49], and yet unidentified tissue-detergent interactions [3,19,49] most likely influence the extent of this ‘‘safety window.’’ Some authors suggest that when DC is used alone (without PC) concentrations of the detergent be 1% [3] as DC > 1% is associated with intense pain, profound tissue necrosis, and prolonged nodularity [2,41,43]. Correct injection depth of PC/DC or DC formulations is also critical [2,21,48,49]. Superficial placement may produce cutaneous breakdown and ulceration [2,48]. However, reports of scarring and ulceration are associated with non-physician injectors, large injection volumes, unverified dosages of unknown medications, and other unorthodox practices [8,13,48,49]. As with other injectables, injection technique and practitioner skill and experience are paramount to safety. Ongoing research should determine the ideal concentration and injection methods to avert adverse sequela while maximizing benefits. PC Revealed The role of PC as an active participant in localized adipolysis had been called into question for years [16,19,32,37,41,45,46]. Some contended that the inclusion of PC in adipolytic therapies is based on historical artifact and untested hypotheses [5,16]. However, ironically, the hypothesis that this phospholipid could not lyse cells or reduce fat was based on indirect evidence (i.e., there appeared to be minimal difference in experimental and clinical outcomes when PC was added to DC). DC could be studied independently of PC because it is water soluble, making the design of experimental studies relatively straightforward. On the other hand, conventional phospholipid solvents like chloroform and ethanol are cell toxic and poor candidates to solubilize PC, making experimental designs to isolate and scrutinize PC troublesome. This quandary was recently solved by Duncan et al. [2], who designed a novel method to investigate the effect of PC on adipocytes using biologically inactive mineral oil. In this way, the isolated effect of PC on cells could be observed without confounding effects from the solvent. The authors incubated human-derived adipocyte stem cells from abdominal fat and induced them to maturity. Cytotoxicity assays (lactate dehydrogenase and oil red O) and lipolysis assays (glycerol and triglyceride assays) were performed on the cultured adipocytes after exposure to PC(5%)/ mineral oil, DC (1% and 2.4%), benzyl alcohol, and isoproterenol (a b-agonist) (Table 2). These data are the first to experimentally confirm that PC has no adipolytic (i.e., fat cell lysing) or lipolytic (i.e., free fatty acid liberating) effects. Investigators and clinicians continue to incorporate PC with DC. While is has been suggested that PC transports DC-lysed cells and their contents away from the treatment site (‘‘lipidic drainage’’) [41], this has not been verified

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TABLE 2. Deoxycholate Containing Formulations Induce Cell Lysis Test solution PC50/DC42 Deoxycholate 1% Deoxycholate 2.4% Phosphatidycholine 5% in mineral oil Isuprel 0.08% injectable Local anesthetic 5% Saline 0.9% (Control) Benzyl alcohol

Adipocyte cell lysis obtained with this solution þþ þþþ þþþ 0 0 0 0 0

PC in isolation from DC does not cause adipolysis (fat cell lysis), nor lipolysis (triglyceride breakdown). These data are the first to experimentally confirm prior deductions that PC will not reduce fat without DC. Obtained with permission from Diane Duncan, M.D. Averaged results with all assays: lactate dehydrogenase, Oil Red-O glycerol, triglyceride.

experimentally. Yet it does appear that PC reduces the morbidity of the procedure by buffering the ablative effects of DC on tissue [2,3,41]. Whereas injections of relatively high concentrations ( > 1%) of DC alone produce profound inflammation, prolonged nodularity, and potentially, skin necrosis [2,37,42,43,45], PC/DC combinations that use higher concentrations of DC (up to 4.75%) appear not as likely to produce these effects [2,41]. These paradoxical observations may be explained by the behavior of aqueous mixtures of DC and PC, which spontaneously form detergent/phospholipid aggregates called micelles [50,51]. It has been conjectured [3] that while unbound DC is capable of ablating tissue, PC/DC aggregates will not. Therefore, the adipolytic effects of PC/DC combinations may be due to unbound DC. A recent double blind study investigating submental fat reduction demonstrated no differences in efficacy or safety of a low concentration (1%) DC formulation compared to a PC (5%)/DC (4.75%) formulation. Using 1% DC appears to be a reasonable approach for clinicians who favor a single agent formulation [3,43,44]. As with most drugs, a safe and effective single ingredient medication is more desirable than a multi-ingredient formulation, unless the latter possesses significant advantages. CONCLUSIONS A simple, safe and effective injectable capable of reducing fat may become available in the near future. Injectable fat reducing therapies are not an alternative to liposuction. Rather, they may be best suited for patients unwilling or unable to have surgical reduction of small collections of fat, or for those patients who desire touch-ups for liposuctioninduced irregularities. Extensive pre-clinical safety testing and rigorous clinical trials demonstrating a favorable product profile using a pharmaceutical grade formulation will be required for regulatory authority approval.

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