PRUEBAS DE EFICACIA DE CRESCINA

Test clínico - Clínica Dermatológica Hospitalaria.

Fecha: 15 de julio de 1999

Estudio clínico que demostró la eficacia de Crescina como coadyuvante del crecimiento natural del cabello y como preparado capaz de reducir la raleza del cuero cabelludo. El estudio fue realizado por el equipo médico de la Clínica Dermatológica en una estructura hospitalaria pública durante el julio de 1999.
Consistió en 6 meses de tratamiento con 30 voluntarios: 19 hombres y 11 mujeres de 20 a 52 años. Los hombres presentaban raleza hasta el grado V de la escala de Hamilton y las mujeres hasta el grado III de la escala de Ludwig. En el lado derecho del cuero cabelludo se aplicó la loción Crescina y en el lado izquierdo una loción placebo.
A los 2 meses de tratamiento, la clínica verificó un re-crecimiento del lado del cuero cabelludo tratado con Crescina y lo consideró bueno en 13 personas sobre 30, satisfactorio en 13 sobre 30 y modesto en 4 sobre 30. Es decir que, a los 2 meses de tratamiento, todas las personas tratadas habían experimentado un resultado positivo. En el 86,6% el resultado se consideró entre satisfactorio y bueno.

A los 4 meses, el estudio demostró un re-crecimiento que fue considerado:
- bueno en 21 personas sobre 30
- satisfactorio en 7 personas sobre 30
- modesto en 2 personas sobre 30.

Es decir que, después de 4 meses, el re-crecimiento resultó entre satisfactorio y bueno en el 93,3% de las personas tratadas.

A los 6 meses, los resultados fueron los siguientes:
- re-crecimiento bueno en 25 personas sobre 30
- re-crecimiento satisfactorio en 5 personas sobre 30.

Esto significa que todo el panel de voluntarios experimentó resultados entre satisfactorios y buenos después de 6 meses. En la parte del cuero cabelludo tratada con placebo, el resultado fue clasificado como nulo o modesto.
Pero el estudio clínico evaluó no sólo el re-crecimiento sino también la resistencia y el trofismo del cabello y el estado de raleza.
- Resistencia del cabello a los 6 meses de tratamiento: buena en 27 personas sobre 30, satisfactoria en 3 personas sobre 30.
- Trofismo del cabello a los 6 meses de tratamiento: bueno en 28 personas sobre 30, satisfactorio en 2 personas sobre 30.
La evaluación de la raleza merece una explicación por separado, no sólo por su importancia para los pacientes sino también por la diversidad de las condiciones basales.

Juicio final general expresado por el Director de Dermatología a los 6 meses de tratamiento con respecto al estado de raleza: bueno para 21 personas, satisfactorio para 9 personas.
Cabe notar que el 50% de la población de la muestra notó mejorías del estado de raleza tras sólo 2 meses de tratamiento.

Test clínico-instrumental - Instituto de Investigaciones Químicas y Bioingeniería (Milán)

Fecha: 5 de mayo de 1999

Este test, que se tituló “Estudio clínico de evaluación de la actividad de una loción tricológica (Crescina) en personas afectadas por raleza debida a alopecia androgenética y/o defluvium” se realizó con un grupo de 25 voluntarios, 14 mujeres y 11 hombres, de 23 a 45 años afectados por alopecia androgenética de grado I-II de la escala de Ludwig en el caso de las mujeres y de grado II-III de la escala de Hamilton en el caso de los hombres, y/o afectados por defluvium, en buen estado de salud y anuentes. Tres voluntarios decidieron abandonar el tratamiento, por lo que el estudio se llevó a cabo con 22 de los 25 pacientes iniciales.

Test clínico-instrumental - Instituto de Investigaciones Químicas y Bioingeniería (Milán)

Fecha: 20 de febrero de 2001.

Este test, que se tituló “Estudio clínico de evaluación de la actividad de una loción tricológica (Crescina) en personas afectadas por raleza debida a alopecia androgenética y/o defluvium”, se realizó con un grupo de 42 personas que utilizaron el producto Crescina en el ámbito de un estudio comparativo más amplio, en el que participaron un total de 84 personas, entre Crescina y un producto de referencia, aplicado con las mismas modalidades. Aplicación: loción aplicada directamente (2,5 ml) a las partes más ralas del cuero cabelludo, friccionando ligeramente hasta la absorción total.
En condiciones basales (T0), a los dos (T2) y a los tres meses (T3) se realizó un fototricograma.
Además, para cada uno de los tres campos (0, 2, 3) se fotografió un área alopécica. Población de muestra: 42 voluntarios (24 mujeres y 18 hombres). Afectados por distintos grados de alopecia: I-II de Ludwig las mujeres, II-III-IV-V de Hamilton los hombres. 5 personas abandonaron el tratamiento. Las personas restantes en el protocolo fueron 37.

- Resultados del test
Fototricograma de personas con diagnóstico de defluvium.

- Conclusiones
Crescina desempeña una actividad trófica, es decir, estimula el crecimiento del cabello en personas afectadas por raleza de distinta naturaleza.
En personas con diagnóstico de defluvium: mejoría a los dos meses de tratamiento. En personas con diagnóstico de alopecia: aumento de la cantidad de cabello en fase anagen con respecto al número en fase telogen/catagen. Efecto demostrado tanto en mujeres como en hombres.

EJEMPLOS DE FOTOGRAFÍAS DE LOS ESTUDIOS

ESTUDIO CLÍNICO DE EVALUACIÓN DE LA ACTIVIDAD DE UNA LOCIÓN TRICOLÓGICA CON PERSONAS AFECTADAS POR RALEZA DEBIDA A ALOPECIA ANDROGENÉTICA Y/O DEFLUVIUM - DERMING, INSTITUTO DE INVESTIGACIONES CLÍNICAS Y BIOINGENIERÍA.
FOTOGRAFÍAS DE LA CUENTA ELECTRÓNICA DEL CABELLO

EJEMPLOS DE FOTOGRAFÍAS DE LOS ESTUDIOS

ESTUDIO CLÍNICO DE EVALUACIÓN DE LA ACTIVIDAD DE UNA LOCIÓN TRICOLÓGICA CON PERSONAS AFECTADAS POR RALEZA DEBIDA A ALOPECIA ANDROGENÉTICA Y/O DEFLUVIUM - DERMING, INSTITUTO DE INVESTIGACIONES CLÍNICAS Y BIOINGENIERÍA.
FOTOGRAFÍAS DE UN ÁREA ALOPÉCICA (LÍNEA DE INSERCIÓN DEL CABELLO EN LA REGIÓN FRONTAL-TEMPORAL DERECHA)

"IN VITRO" TEST

Effects of Crescina® on human primary epidermal and dermal cells -
Investigators: V C Biotechnology

Aim of project
It has been tested a preparation formed by the nucleus of Crescina hair treatment active principles in an aqueous solution and with the following composition: aqua, glycerin, lysine hydrochloride, acetyl cysteine, glycoproteins.

The objectives of this project were the following:

Objective 1
We investigated effects of Crescina on human primary keratinocytes and fibroblast cell proliferation

Objective 2
We investigated the effects of Crescina on protein synthesis in human in primary human keratinocytes and fibroblasts.

Abbreviations used
NHEK: Normal human epidermal keratinocytes
NHDF: Normal human dermal fibroblasts
SD: Standard deviation
MV: Mean value

Study protocol

Objective 1

Cell Cultures. Primary human keratinocytes and fibroblasts were obtained from Promocell (Heidelberg, Germany). Cells were maintained in specific growth medium supplemented with growth factors and seeded in 96 well plates (10 000 cells/well) for determination of cell proliferation and in 24 well plates for protein synthesis. Cells were treated for different period of time (24, 48 and 72 hours) with increasing concentrations (0.031, 0.062, 0.125, 0.25, 0.5% w/v = 310 µg/ml, 620.5 µg/ml, 1.25 mg/ml, 2.5 mg/ml, 5 mg/ml provided as 0.31 µl, 0.62 µl, 1.25 µl, 2.5 µl, 5 µl) of a solution of Crescina. Medium was removed before stimulation and replaced by fresh medium without growth factors. The growth factor supplements, as well as the growth factors PDGF (platelet derived growth factor) and FGF (fibroblast growth factor), were used as positive controls. Not-treated cells have been used as negative control.

Determination of cell proliferation.
Fibroblasts or keratinocytes were incubated with Crescina for 24 h, 48 h, and 72h. Each dose (see above) was tested 16 times (8 wells on 2 plates with two different methods).
Proliferation of primary fibroblasts/keratinocytes was determined by:

Determination of protein content.
Human primary keratinocytes or fibroblasts from human skin origin were seeded in 24 or 6 well plates and incubated with the Crescina (5 doses see above) for different times (24, 48 and 72 hours) with n = 4. After cell treatment, cells will be washed with phosphate buffered saline (PBS) and lysed in 1.3 x SDS (sodium dodecyl sulfate)-containing sample buffer without DTT containing 100 µM orthovanadate (Laemmli, 1970). Lysates will be homogenized by repeated passage through a 26-gauge-needle. Protein contents will be measured using the bicinchoninic acid method (BCA protein determination kit from Pierce, distributed by KFC Chemikalien, München, Germany) according to the manufacturer’s instructions (standards (bovine serum albumine, Sigma) ranging form 0.2 µg/µl to 4 µg/µl; optical density read at 570 nm). This method is a well established method to determine protein content and was part of at least 20 high impact publications from our group (e.g. Akundi et al., 2004).

Deviations:
To obtain more valid data in fibroblasts, additional experiments were performed using less cell numbers (1 000 and 5 000), and two additional time points.

Results and Discussion

Determination of cell proliferation.
On human keratinocytes experiments, at all tested time points 24, 48 and 72 h, Crescina had no effect on DNA synthesis as measured by dBrdU incorporation.
In contrast, Crescina revealed to increase the reaction of AlamarBlue. This effect was significant, suggesting that Crescina has an effect on cell viability (underlining values > 100% of metabolic activity) compared to non-treated cells (negative control) which cell vitality was considered as 100%. The effects were especially evident with 0.5% of the product Crescina with a maximum value of + 55% at 24 h (Fig. 4) and with 0.25% of the product Crescina with maximum values of + 56.3% at 48 h (Fig. 5) and + 61.6% at 72 h. Furthermore, our data suggest, that Crescina increases the metabolic activity of cells by increasing cellular energy levels.

At all tested time points fibroblastic DNA synthesis was not affected by Crescina, even if it’s possible to notice a quite moderate effect on DNA synthesis as measured by dBrdU incorporation (maximum value + 5.1% with 0.25% of the product Crescina after 48 h) in comparison with non-treated cells.
In the fibroblast experiments using the AlamarBlue method, additional experiments were performed using an inferior number of cells (1 000 and 5 000 cells/well) and two additional time points (4 and 6 h) just to obtain data of major validity. The use of 10 000 cells as starting point did not gain significant data since the fibroblasts are rapid proliferating cells and therefore cell proliferation and metabolism was probably maximal and not further increasable with this amount of cells suggesting that cell metabolism was already decreased due to cell to cell interaction lowering the proliferation rate of the cells.
In the AlamarBlue assays with 1 000 cells/well, Crescina showed a quite moderate increase of metabolic activity, compared to the negative control, after 4 and 6 h with maximal values of + 14.5 % and + 20.5% respectively, both with 0.5% of Crescina.
Very potent effects were obtained after 24, 48, and 72 h (Figs. 13-14) with maximal increase in the metabolic activity of + 58.5% with 0.125% of Crescina at 24 h, + 111.4% with 0.25% of Crescina at 48 h and + 195.7% with 0.5% of Crescina at 72 h, compared to negative control.
Furthermore, in the AlamarBlue assays determining the Crescina effects on cell proliferation with 5 000 cells/well, Crescina showed an increase in the metabolic activity, compared to non-treated cells (negative control), 4 and 6 h with maximal values of + 33.1% and + 36.5% both with 0.5% of Crescina.
Very potent effects were detected after 24, 48, and 72 h (Figs. 18-19) showing an increase in metabolic activity of + 80.3% with 0.0625% of Crescina at 24 h, + 119.3% with 0.5% of Crescina at 48 h, and + 91.4% with 0.5% of Crescina at 72 h, all of them compared to negative control.
In this way, Crescina increased AlamarBlue reactivity when 5 000 cells were seeded but not as potent as with 1 000 cells per well.
Only slight effects were visible after 24 h, when 10 000 cells where seeded (+ 20.2% with 0.5% of Crescina), and no effects after 48 and 72 h (Fig. 20), suggesting that the proliferation and the viability of cells is not further increasable after later time points. This indicated, that the number of cells is critical for obtaining effects of Crescina and that the number of 10 000 cells is to high to induce cell viability by possible cell to cell interactions occurring in confluent cell layers.

Determination of protein content.
Determination of protein content performed using keratinocytes revealed that, compared to non-treated cells, Crescina increased protein content (+ 160.3%) at the dose of 0.125% of Crescina at 24 h, at the doses of 0.0625% (+ 242.1%) and 0,125% (+ 206.8%) after 48 h and, at least, after 72 h with 0.25% of Crescina it’s possible to notice an increase of protein content of + 73.9% (Fig. 23, 24).
No increase in protein content was visible in fibroblasts after 24 h, but a slight increasing effect after 72 h: + 51.4% with the exposition at 0.125% of Crescina.

Conclusions

Determination of cell proliferation.
Using human keratinocytes, the preparation Crescina had no effects on DNA synthesis as measured by dBrdU incorporation at all tested time points 24, 48 and 72 h.
However, Crescina increased the metabolic activity of cells by increasing cellular energy levels as revealed by the reaction of AlamarBlue.
Using human fibroblasts, Crescina had no effect on DNA synthesis as measured by dBrdU incorporation at all tested time points 24, 48 and 72 h.
In the AlamarBlue assays, very potent effects were obtained after 24, 48, and 72 h with an increase in the metabolic activity compared to non-treated cells .
Our data suggest that due to its positive effects on cell viability and metabolism as shown by the AlamarBlue assays, Crescina should also increase the cell number. Furthermore, we conclude that the treatment with Crescina increases cell life, finally resulting in increased cell numbers, if compared to untreated cells, which have a more rapid cell turnover. This increase in cell numbers in Crescina treated cells, will not increase DNA synthesis as observed in this study.

Determination of protein content.
Determination of protein content in keratinocytes revealed that Crescina increased protein content considerably after 24 h (+ 160.3%) and 48 h (+ 242.1%) compared to negative control.
In fibroblasts, a slight increasing effect was visible after 72 h (+ 51.4%) compared to non-treated cells.
The difference in the total protein content between keratinocytes and fibroblasts is due to the differences in the total cell volume between the epithelial keratinocytes and the smaller fibroblasts.
Therefore, the protein content has not increased in consequence of the pro-proliferative effect of Crescina, but it might be increased due to increased cell viability. This fits to the hypothesis that Crescina promotes cellular metabolism.

Finally, we demonstrated that preparation Crescina increased cell metabolism and therefore might act as a cell protective and energizing hair treatment.

Figures

LAS RESPUESTAS DEL MERCADO

Labo ha considerado correcto y necesario para su investigación, su publicidad y su política comercial interpelar al mercado con el objetivo de conocer la opinión de la clientela primaria (farmacéuticos) y final (consumidores) sobre la calidad del producto y su grado de satisfacción. Los resultados de las encuestas han sido sumamente positivos.

A) Testimonios de eficacia de los farmacéuticos

Labo ha recogido testimonios directos de los farmacéuticos sobre la calidad del producto Crescina y sobre la satisfacción de la clientela. La muestra es significativa: 497 farmacias de todo el territorio nacional.
Las farmacias encuestadas timbraron y suscribieron el siguiente cuestionario: