Human Placental Protein

HUMAN PLACENTAL PROTEINS, LIPIDS, AND EXTRACTS 87

Using the extraction technique of Florini et al. (1966), Riggi et al. (1966) reported that intramuscular (IM) administration of “puriŽ ed human placental protein” into fasted rabbits (25 or 50 mg/kg), and monkeys (50 or 100 mg/kg) produced signiŽ cant increases in plasma free fatty acid concentrations. Plasma lactescence associated with hypertriglyceridemia and hyperglycemia developed in rabbits following daily SC dos-ing with 75 mg/kg for 25 days. In mice, hepatic lipidosis de-veloped after injections with 16 mg of Human Placental Pro-tein daily for 7 days. All of the described effects were similar to those observed following porcine growth hormone ad-ministration.

Human Placental Extract (presumed active, see Introduction)

Banerjee, Bishayee, and Chatterjee (1993) reported that a single IP dose of HPE (4 ml/kg) to rats caused a signiŽ cant en-hancement of lipid peroxidation with a decline in both hepatic and blood glutathione (GSH) concentrations. A dose-dependent increase in glutathione S-transferase (GST) activity and dose-dependent inhibition of catalase, glutathione peroxidase, and glutathione reductase activities were noted. The extract was considered hepatotoxic because it increased serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transami-nase, serum lactate dehydrogenase activities, and blood methe-moglobin concentration. The magnitude of the increase of serum enzymes was “much less” than that induced by carbon tetrachloride.

In a subsequent study by Bishayee, Banerjee, and Chatterjee (1995), rats were given a single IP injection of HPE (4 ml/kg) and some were killed at 2, 6, 24, 45, 72, and 96 hours post treatment. Livers were removed, homogenized, and centrifuged, and the fractions were analyzed for cytochrome and enzymic activity. The vehicle control received 1.5% (v/v) Benzyl Al-cohol in buffer. Maximal induction of hepatic microsomal cy-tochrome P-450 and cytochrome b5 activities was noted begin-ning at 24 hours post dosing. Cytosolic GST activity was also signiŽ cantly increased beginning at 48 hours post dosing. A re-duction in microsomal UDP-glucuronyltransferase activity was also observed. All activity returned to zero-time values 96 hours after treatment.

Parenteral Toxicity—Short-Term

Human Placental Extract (presumed active, see Introduction) Dose-dependent increases in hepatic cytochrome parameters

and GST activity were noted in rats following repeated IP dosing (30 days) with 1, 2, or 4 ml/kg HPE. The cytochrome changes were signiŽ cant with the 2 ml/kg dose ( p < :05); more pro-nounced increases were noted in the 4 ml/kg dose group where the change in cytochrome P-450 activity was 130% ( p < :01) and the increase in cytochrome b5 activity was 88% ( p < :05) greater than the control. Microsomal NADPH cytochrome c re-ductase activity was not affected by either acute or repeated treatment. The investigators cautioned that human placental ex-tract had “substantial ability to alter the patterns of drugs me-

tabolizing enzyme systems in mammals,” and that prolonged administration could induce some forms of hepatic neoplasms (Bishayee, Banerjee, and Chatterjee 1995).

Similar Ž ndings were reported in an earlier study in which HPE (1 to 4 ml/kg) was injected IP into rats for 15 days. Sig-niŽ cant increases were noted in the activities of serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, lac-tic dehydrogenase, alkaline phosphatase, glutamic dehydroge – nase, and sorbitol dehydrogenase. Activities of other enzymes were also increased. A marked depletion of cytochrome P-450 and reduction of hepatic glycogen and protein concentrations were noted with a concurrent rise in hepatic lipid peroxides. The investigators considered that the active components of the extract, 19-hydroxyprogesteron e and corticotropin-releasing factor, were responsible for the alterations (Banerjee et al. 1994a).

Dermal Irritation

Human and Animal Placental Extract (presumed active, see Introduction)

Two Filatov-type placental extracts (human and bovine) used for “dermocosmetology” were each applied (0.5 ml) under gauze to rabbit skin. Both extracts were nonirritating. No further details were given (CTFA 1998).

Ocular Irritation

Human and Animal Placental Extract (presumed active, see Introduction)

Two Filatov-type placental extracts (human and bovine) used for “dermocosmetology” were each instilled (0.5 ml) into one conjunctival sac of six rabbits. The human placental extract was “very slightly irritating” and the bovine placental extract was nonirritating. No further details were given (CTFA 1998).

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY In Vitro

Animal Placental Extract (presumed active, see Introduction) Mammalian embryonic development was studied by

Huxham et al. (1982) using a postimplantation rat-embryo cul-ture. Wistar rat conceptuses were explanted on day 9.5 and cul-tured for 2 days with homogenate preparations from normal rat placenta or decidua. Conceptuses were examined for heart beat, vitteline circulation, yolk-sac diameter, and the achieve-ment of allantoic fusion with the ectoplacental cone. Abnor-mal embryos (neural-tube defects, severe reduction in embry-onic size) were produced with 2.5 to 4 mg/ml of the placental homogenate and 1.2 to 4 mg/ml of the decidual homogenate. Abnormalities were not induced by either solutions of bovine serum albumin or protein preparations of rat lung tissue.

88 COSMETIC INGREDIENT REVIEW

GENOTOXICITY

Human Umbilical Extract

Immunizing mice with an extract of human umbilical cord signiŽ cantly decreased the number of micronuclei in bone mar-row cells for 5 days. The extract was described as having no clastogenic property. The investigators hypothesized that the antimutagenic effect was related to interferon induction by the extract (Mkrtchian and Nersessian 1993).

PROTECTIVE ACTIVITY

Human Placental Extract

Klein et al. (1991) reported that in in vitro studies, EAP in-hibited growth of Ha-ras– transformed BALB/c 3Tc cells and human squamous lung carcinoma A-2182 cells. The fraction did not alter anchorage-dependent growth of these cells, but a slight mitogenic activity was noted in nontransformed cells. No sig-niŽ cant cytotoxicity was noted. The fraction did contain trans-forming growth factor ¯, but the investigators did not consider that the growth factor was solely responsible for the observed growth suppression.

In a subsequent study, Klein, Chiodino, and Yamasaki (1993) reported that EAP suppressed growth of only the most highly tumorigenic cells in soft agar medium; growth of non- and low-tumorigenic counterparts was not affected or was stimulated, respectively, by the extract. Cells of both the colorectal and esophageal cell lines that had the greatest percentage of colonies in soft agar had their colony-forming efŽ ciency decreased by the presence of 100 ¹g/ml EAP. In contrast, cells that did not give any colonies in soft agar did not grow in either the absence or presence of EAP. Growth of cells with an intermediate colony-forming efŽ ciency was stimulated (by 150% in colorectal cells, and 200% in esophageal cells) in the presence of EAP. Similar

Ž ndings were noted with murine BALB/c 3T3 1-1 cells that had been transfected or infected with various oncogenes. Further, “EAP did not signiŽ cantly affect the doubling time of anchorage-dependent cell growth, suggesting that the extract speciŽ cally suppresses tumorigenic characteristics of cells such as their abil-ity to grow in soft agar medium.” Transforming growth factor ¯ was most effective on less tumorigenic cells.

Human Placental Extract (presumed active, see Introduction) Komura et al. (1983) conducted a chemical study on the antimutagenic action of human placental extract. A human pla-centa was washed, diced, and homogenized. The liquid was centrifuged; then the supernatant was boiled and recentrifuged, it was a pale pink liquid. The yield from one placenta was »300 ml of “active juice.” Two placentas were used to prepare HPE1 and HPE2. Bacteria (Eschericheria coli) were mutated by either radiation or incubation with a chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and then com-bined with HPE and plated. A speciŽ c activity AD50 was de-Ž ned as the dose that reduced the number of induced mutations

by 50% without affecting cellular survival. The speciŽ c activities of HPE1 against ultraviolet (UV)-, ° -ray–, and MNNG-induced mutations were 160, 50, and 200 ¹l/plate, respectively. The spe-ciŽ c activities of HPE2 against UV- and MNNG-induced muta-tions were 50 and 80 ¹l/plate, respectively. Cobalt(II) ions were considered essential for the antimutagenic action. However, the investigators noted studies using other mammal placentas and considered that “low molecular, nonproteic factors” could also have played a role.

Placental Extract (presumed active, see Introduction) Mochizuki and Kada (1982) investigated the antimutagenic

action of extracts prepared from the placentas of a human, mon-key, dog, rat, and mouse. Placentas were washed with potas-sium chloride, homogenized without buffer, and centrifuged. The supernatant was treated with pronase followed by overnight dialysis in distilled water. Each solution was heated and loaded onto ion-exchange resin columns and eluted with water. Frac-tions were collected and evaporated under vacuum; the residue was dissolved in water and Ž ltered using millipore Ž lters. Bac-teria (E. coli B/r WP2 trp¡) were mutated by either radiation or incubation with MNNG and then combined with an extract and plated. The extracts were also tested alone and were not mutagenic. The number of mutant colonies induced by UV irradiation, ° -ray, and MNNG were “decreased markedly in the presence of the placental extracts without signiŽ cant ef-fects on survival.” The data were not analyzed for statistical signiŽ cance.

Human Umbilical Extract

Vaccination of rats and mice with an extract of human umbil-ical cord resulted in a signiŽ cant inhibition of growth, decreased tumor incidence, and partial resorption of ascitic • uid of trans-plantable tumors such as Ehrlich’s ascites tumor, sarcoma 37, and Zajdel’s hepatoma. The inhibition was not noted when sar-coma 180 was transplanted. Vaccination also interfered with dimethyl benzanthracene and benzo(a)pyrene-induced carcino-genesis by reducing tumor incidence and increasing the latent period and slowing cancer progression (Mkrtchyan et al. 1990).

CLINICAL ASSESSMENT OF SAFETY

Patch Testing

Human Placental Protein

A patch testing reference book by DeGroot (1994) noted that the published literature does not contain recommended test con-centrations for Human Placental Protein. As a guide to the clin-ician, DeGroot reported the Ž ndings of an unpublished (and at the time, ongoing) study by members of the Dutch Contact Dermatitis Group. No irritant reaction was noted in 1 to 20 pa-tients (exact number tested with ingredient not speciŽ ed) suffer-ing from or suspected to suffer from cosmetic product contact al-lergy after being patch tested with 30% Human Placental Protein aqua.