阅读说明：本技术 表皮生长因子在治疗糖尿病性足溃疡中的用途 (Use of epidermal growth factor in the treatment of diabetic foot ulcers ) 是由 A·德里奥马丁 J·E·萨屋里察维兹 R·乌比塔戈麦斯 S·冈萨雷斯巴兰寇 J·A·贝尔兰嘉 于 2020-03-16 设计创作，主要内容包括：本发明涉及表皮生长因子(EGF)在制备可注射药物中的用途,所述可注射药物用于在评价之时不具备患病肢截肢标准的患者中治疗糖尿病性足溃疡(DFU),其中每周一次通过损伤内浸润到所述溃疡中来施用所述包含EGF的药物。此外,本发明提供了用于在患者中治疗DFU的包含EGF的药用组合物,其中每周一次施用所述组合物。本发明还考虑了用于在评价之时不具备患病肢截肢标准的患者中治疗DFU的方法,其包括每周一次通过损伤内浸润到所述溃疡的床体中来向所述患者施用EGF。(The present invention relates to the use of Epidermal Growth Factor (EGF) in the preparation of an injectable medicament for the treatment of Diabetic Foot Ulcers (DFU) in patients not having criteria for affected limb amputation at the time of evaluation, wherein said medicament comprising EGF is administered once a week by intralesional infiltration into said ulcers. Further, the present invention provides a pharmaceutical composition comprising EGF for use in treating DFU in a patient, wherein the composition is administered once weekly. The invention also contemplates a method for treating DFU in a patient who does not have the criteria for diseased limb amputation at the time of evaluation, comprising administering EGF once per week to said patient by intralesional infiltration into the bed of said ulcer.)

1. Use of Epidermal Growth Factor (EGF) in the manufacture of an injectable medicament for the treatment of Diabetic Foot Ulcers (DFUs) in patients not having criteria for diseased limb amputation at the time of evaluation, wherein said medicament comprising EGF is administered once a week by intralesional infiltration into the bed of said ulcers.

2. The use of claim 1, wherein the amount of EGF in each dose of medicament administered to the patient is from 2 to 80 μ g.

3. Use according to claim 1, wherein the assessment of DFU is performed at the start or during the course of the treatment.

4. The use of claim 1, wherein the DFU is treated three times per week with EGF administered by an intralesional route prior to once per week treatment by intralesional infiltration into the bed of said ulcer with EGF.

5. The use of claim 1, wherein the patient is treated with conventional therapy during the same course of treatment.

6. A pharmaceutical composition for treating Diabetic Foot Ulcer (DFU) in a patient not having the criteria for diseased limb amputation at the time of evaluation comprising Epidermal Growth Factor (EGF) and a pharmaceutically acceptable excipient, wherein the composition is for once weekly administration.

7. The composition of claim 6, wherein the amount of EGF in each dose administered to the patient is from 2 to 80 μ g.

8. A method of treating a Diabetic Foot Ulcer (DFU) in a patient who does not have the criteria for diseased limb amputation at the time of evaluation, comprising administering a therapeutically effective amount of Epidermal Growth Factor (EGF) to the patient once a week by intralesional infiltration into the bed of the ulcer.

9. The method of claim 8, wherein the amount of EGF in each dose administered to the patient is 2 to 80 μ g.

10. The method of claim 8, wherein the assessment of DFU is made at the beginning or during the course of treatment.

11. The method of claim 8, wherein the DFU is treated three times per week with EGF administered by an intralesional route prior to once per week treatment by intralesional infiltration into the bed of the ulcer with EGF.

12. The method of claim 8, wherein the patient is treated with conventional therapy during the same course of treatment.

Technical Field

The present invention is in the field of human medicine and pharmaceutical industry. In particular, the present invention relates to the use of Epidermal Growth Factor (EGF) in the manufacture of a medicament for the treatment of Diabetic Foot Ulcers (DFU). The drug is administered by infiltration into the bed of the lesion within the lesion.

Prior Art

Diabetes is a significant problem for public health services and is one of the most common non-infectious chronic diseases. The international diabetes association proposed that this disease has a global prevalence of 8.3% among adults. According to the statistics of the world health organization, it is expected that the number of diabetics will exceed 6.29 million by 2045 years (International Diabetes mellitus Federation. the global bureden. in: International Diabetes mellitus Federation. IDF Diabetes atlas. geneva: IDF,2017 p.9-50). Currently, diabetes continues to show accelerated growth, mainly due to poor eating habits and lack of systematic implementation of exercise, which has made it a non-infectious disease with a world pandemic scale.

Education of treatment for diabetics is the basis for their treatment. Self-care measures and lack of regular out-patient follow-up are not known to favor the emergence of complications such as DFU (Al-Wahbi am. vasc Health rise manag.2010,6: 923-34; dorrestein J, Valk jd. diabetes Metab Res Rev 2012,28(Suppl 1): 101-6). Although prevention programs have been enacted and health services organized, amputation remains an important problem to be solved (Garcia a Y et al, Revista Cubana de angiologia y circlia Vascular 2016,17(1) 36-43).

The concept of a Diabetic Foot is difficult to define accurately (Apelqvist J et al, International Working Group on the Diabetic Foot edition Board. diabetes metals Res Rev 2008,24(Suppl 1): S181-7). In the 2007 literature of the International clinical guidelines for Diabetic feet (International Consensus on the Diabetic Foot), a Diabetic Foot is defined as "an ulcer, infection or destruction of deep tissues associated with neuropathy and/or peripheral arterial disease in the lower limbs of a person with diabetes". A foot is considered to be at risk when certain factors are present that predispose the foot to ulceration, such as neuropathy, ischemia, deformity, callus and edema (Garcia Herrera AL, Febles Sanabria R, Moliner Cartaya M. Revista Cubana de angioplastic i a Ciruga Vascal 2016,17(1): 13-24).

For the treatment of DFU there are different types of treatments in the prior art, approved by international consensus and published in journals of great influence, such as especially the National Institute for Health and Care Excellence of the United Kingdom (NICE). Among these, pressure-site discharge, vacuum therapy, hyperbaric oxygen administration, dressings with various characteristics (which are applied according to the type of injury) and broad-spectrum antimicrobial agents (alone or in combination) may be indicated. There are also different types of debridement, whether mechanical, biological, or chemical. Furthermore, skin substitutes were applied in these patients (https:// nice. org. uk/guidance/ng19, publication date: 8 months 2015). Nevertheless, none of these treatments have been able to reduce the high percentage of amputations performed annually due to DFU. The healing of ulcers is the most important event for the patient. However, in lesions that do not respond to conventional or standard treatments, granulation (as an intermediate and related event for lesion closure) is of great significance because it reduces the risk of ulcer infection and diseased limb amputation.

Since the beginning of the 90 s, the administration of growth factors was evaluated in the treatment of chronic wounds. One of these growth factors is EGF, a low molecular weight protein recognized since the 50 s of the last century (Hardwicke J et al, Surgeon 2008,6: 172-177). The growth factor was repeatedly applied by the topical route during administration trials in chronic wounds, venous ulcers and DFU.

The microenvironment in diabetic chronic wounds is hostile to the stability of local growth factors, their chemical integrity, their bioavailability and their physiological role (as the main driver of the healing process). Within this environment, the receptor is persistently expressed and its signaling ability is impaired (Berlanga-Acosta J et al, Int Wund J2013, 10: 232-.

It is known that the improvement of wound healing by EGF is promoted under a prolonged, sustained and slow release system, which causes its receptor to be constantly expressed. According to the prior art, there is a need for constant exposure of EGF to its receptor. This means that EGF-related wound healing properties can be manifested if the receptor is exposed and constantly occupied for at least 8 to 12 hours (Knauer DJ et al, J Biol Chem 1984,259: 5623-.

On the other hand, prolonged local bioavailability and timely stimulation of receptors have been demonstrated to be required to produce significant effects mediated by EGF in Wound closure (Kasayama S, Ohba Y, Oka T, Proc Natl Acad Sci U S A1989, 86: 7644-.

A lyophilized product for intralesional use having recombinant human egf (rhegf) as an active ingredient is registered as a medicament for the treatment of DFU. The intralesional application of the drug provides recombinant proteins to the bed of ulcers (replacement therapy) because cells of the injured microenvironment are unable to synthesize endogenous proteins, or are synthesized in low proportions. This deficiency interferes with the healing of ulcers, which leads to chronicity and, therefore, the appearance of complications such as local infections and diseased limb amputations. The drugs mentioned reduced amputation rate by 70% or more, with the greatest percentage reduction among those of less complex ulcers. Intralesional application of rhEGF showed a positive profile of benefit. Several thousand patients with DFU have been treated with injectable rhEGF administered by this route.

rhEGF infiltration at the bottom and edges of DFU was performed in a treatment regimen of three times per week and 75 μ g factor/drug dose administered. From this application, senescent cells (especially fibroblasts and keratinocytes) are awakened, which initiates a change from a chronic pro-inflammatory environment to a physiological reparative environment in the ulcerated area. This ensures proper granulation or useful living tissue, and subsequent lesion closure. This granulation process allows for more than 80% correlation with lesion closure. Although there is a high correlation between granulation and closure, these percentages can vary when patients with moderate ischemia of DFU-affected limbs are included in the treatment. The administration regimen of injectable rhEGF three times a week and at 75 μ g/applied dose has the disadvantage of frequent adverse events and high treatment costs and patient discomfort (which may lead to its withdrawal from the therapy).

Therefore, there is a need to identify new injectable rhEGF administration regimens that allow for correct granulation and effective lesion closure in patients suffering from diabetic foot, while improving the comfort of the individual and its compliance with the treatment.

Detailed Description

The present invention solves the above-presented problems by disclosing the use of Epidermal Growth Factor (EGF) in the preparation of an injectable medicament for the treatment of DFU in patients not having the criteria for diseased limb amputation at the time of evaluation, wherein said medicament comprising EGF is administered once a week by infiltration into the bed of said ulcer within the lesion. In one embodiment of the invention, the amount of EGF in each dose of the medicament administered to the patient is 2 to 80 μ g.

To practice the invention, the evaluation of DFU can be performed in the patient at the beginning (i.e., when making an initial diagnosis) or during the course of treatment to avoid amputation of the ulcerated diseased limb. When the criteria for amputation are considered to be absent, the drug containing EGF may be applied once a week by intralesional infiltration into the bed of the ulcer. In the present invention, patients are treated with conventional therapy prescribed by medical authorities while they receive weekly intralesional infiltrations of EGF into a bed of DFU.

In one embodiment of the invention, the patient has previously been treated to avoid amputation of an ulcerated diseased limb. This previous treatment consisted in administering EGF three times a week by the intralesional route. Once DFU was evaluated and it was confirmed that there were no diseased limb amputation criteria, one switched to treatment once per week by intralesional infiltration of EGF into the bed of the ulcer.

To date, EGF has been administered by an intralesional route to thousands of DFU patients in a treatment that considers three weekly administrations of drugs. However, there is no precedent for the efficacy of an administration regime in which EGF is administered once a week, whether during a portion of the treatment period or during the entire course of treatment. The results of achieving effective closure of DFU with this new regimen with a lower frequency of EGF application cannot be predicted from what was described in the previous literature, since the healing mechanisms in diabetic patients are poorly understood, far from what occurs in non-diabetic individuals.

On the other hand, as previously suggested, it is known that the improvement of wound healing by EGF is promoted under prolonged, sustained and slow release systems, and prolonged local bioavailability and timely stimulation of receptors are required for this. With all these prior knowledge reported in the literature of the technical field, it was not expected that after reducing the frequency of application of EGF in DFU, similar results were obtained as achieved with more frequent applications, even in patients without the criteria of suffering from amputation of the limb, as is the case of the present invention. More precisely, it is expected that treatment at intervals like weekly would induce a decrease in the expression of the EGF receptor and thus a negative response to the therapy with this growth factor. For this reason, initial trials attempting to reduce the frequency of application were conducted with a three-fold dose of EGF (225 μ g) which strived to maintain a sufficient amount of the growth factor in the ulcer for a longer period of time. Surprisingly, the residual amount of rhEGF in the ulcer was detected to be the same as with the conventional dose and maintained the same therapeutic effect. Even more surprising, the same satisfactory results were obtained in this type of ulcer with the conventional dose and with only once weekly application.

In the context of the present invention, epidermal growth factor refers to any variant of the EGF molecule that retains its biological activity; for example, a molecule truncated at the C-terminus (Calnan et al, Gut 2000,47: 622-. Human EGF may be obtained by recombinant deoxyribonucleic acid techniques in hosts known to those skilled in the art (rhEGF). Without limiting the scope of the invention, among these hosts are yeasts, such as Saccharomyces (Saccharomyces) or Pichia (Pichia); or bacteria, such as E.coli (Escherichia coli). The polypeptide may also be obtained by chemical synthesis.

For the purposes of the present invention, it is believed that a DFU affected limb does not meet the criteria for amputation when the tissue has the following conditions: exhibits a mild or unobtrusive moderate degree of ischemia; do not present signs of local infection, malodor, bone involvement (e.g., osteomyelitis) caused by secretions; inflammatory signs affecting the general state of the patient, such as edema, fever, redness, pain and loss of functionality in the ulcerated region, are not present, i.e. the patient does not have signs and symptoms of systemic sepsis; does not exhibit extensive muscle necrosis; presents a bottom with good tension, prone to bleeding during the healing process; with sensitivity that is preserved as much as possible, i.e. the patient exhibits discomfort during the healing process.

The term "evaluation of DFUs" is well known to those skilled in the art and occurs in treatment guidelines established by medical institutions and authorities. On the other hand, for the purposes of the present invention, the term "infiltration of EGF" refers to injection at various points of the bed of DFU.

In another aspect, the invention provides a pharmaceutical composition for treating DFU in a patient who does not have the criteria for suffering from amputation of a diseased limb at the time of evaluation, comprising EGF and a pharmaceutically acceptable excipient. The composition is administered once a week. In one embodiment of the invention, the composition comprises an amount of EGF ranging from 2 to 80 μ g per dose administered to the patient. Without limiting the scope of the invention, for the pharmaceutical composition, EGF is formulated with excipients and carriers (which may be, for example, stabilizers, buffers, and compounds that allow lyophilization without affecting the biological activity of the growth factor).

The invention also contemplates a method for treating DFU in a patient who does not have the criteria for diseased limb amputation at the time of evaluation, comprising administering a therapeutically effective amount of EGF once per week to said patient by intralesional infiltration into the bed of said ulcer. The method of the invention allows for treating the patient with conventional therapy during the same course of treatment.

In one embodiment of the method of the invention, the amount of EGF in each dose administered to the patient is from 2 to 80 μ g. In the methods of the invention, the evaluation of the DFU can be performed at the beginning (i.e., at the time of initial diagnosis) or during the course of treatment to avoid amputation of the DFU-afflicted limb.

In a particular embodiment of the method of the invention, the patient is treated three times a week with EGF administered by the same route in the bed of the ulcer, before receiving the intralesional infiltration of EGF on a weekly schedule. This combination treatment regimen, while effective in patients, also allows for a reduction in the likelihood that these patients suffer from the most common adverse events of administering rhEGF within the lesion (e.g., pain and burning, shivering, and chills) by exposing the patients to a lower total dose of rhEGF via this route.

As seen in the present invention, surprisingly, similar granulation results were obtained with the proposed new treatment regimen in patients receiving comparable rhEGF amounts/doses compared to the established application regimen three times a week over 8 cycles.

Detailed description of the embodiments/examples

Example 1 intralesional administration of rhEGF in three treatment regimes of DFU patients

The characteristics of the DFU patients involved and their lesions are presented in table 1. It can be seen that the mean age was above 60 years, varying between 24 and 87 years. There was no difference between patients' sexes. Type 2 diabetes is predominant. The time to progression of diabetes is over 16 years, and the lesions are mostly grade 3 (classified according to Wagner). The patient was assigned one of three treatment regimens, according to the protocol and the dose of rhEGF administered. The area of damage in these three groups was greater than 20cm²。

rhEGF was administered to the patients in groups 1 and 2 three times a week by the intralesional route at 75 μ g/dose or 25 μ g/dose, respectively, in order to affect the margins and bottoms of the lesions. For this purpose, reconstitution and dilution of a vial containing the drug was performed with 5mL of water for injection. The application is performed until 100% of lesion granulation, lesion closure by transplantation, or up to 8 weeks of treatment is achieved.

The drug was administered to patients belonging to group 3 by intralesional route three times a week during the first week (75 μ g/dose). Administration was continued once a week starting at the second week at a dose of 225 μ g. The treatment period was 8 weeks, although complete closure of the lesion if advanced, or less than 1cm if the ulcer reached²Then it may be shorter.

TABLE 1 demographic data of the patients studied

^aSD: standard deviation;^bQR: four-bit pitch

rhEGF obtained and purified from recombinant yeast was used in all groups. The formulated product was lyophilized in vials containing either 25 μ g or 75 μ g of the active pharmaceutical ingredient. The composition further comprises pharmaceutically acceptable excipients, such as stabilizers and pH adjusters.

Patients in the three groups continued to receive conventional therapy according to the treatment criteria for DFU established by the National Angiology Group of Cuba. This includes: unloading the lesion site, metabolic control of the underlying disease with insulin (which replaces the oral hypoglycemic agent), washing the lesion with saline solution and soap, debridement, application of sterile dressing with gauze bandage, and antimicrobial agents, depending on the type of infectious germs present in the lesion.

Area and percentage of granulation of DFU by commercial system (Visitrak) created for these purposes^TM,Smith&Nephew) and a commercial system comprising a grid of clear acetate fibers and a device for measuring damage by area method. The system has been previously validated (Sugama J et al, J Clin Nurs 2007,16(7): 1265-. The outline of the lesion and the area of granulation are delineated on the acetate fibers by using a permanent marker. The results obtained after intralesional administration of the drug in patients with a high risk of amputation of the affected limb can be seen in table 2.

TABLE 2 response to granulation and lesion closure and amputation following intralesional treatment with rhEGF

CR: complete granulation response at 3 weeks (. gtoreq.75% of the lesion area covered by useful granulation tissue). PR^*: granulation response in the 3 week part: (>50% and<75% of the lesion area covered by useful granulation tissue). CR: complete granulation response (100% of the lesion area covered by useful granulation tissue) at the end of treatment.

The results are excellent with respect to granulation at the end of week 3 in patients to whom a dose of 75 μ g was applied throughout the treatment period (three times a week), and in patients receiving an initial dose of 75 μ g (three times a week) followed by a weekly dose of 225 μ g. The values reached in this last group are surprising. At the same measurement point of the ulcer, 64% granulation was seen in the group of patients treated with a dose of 25 μ g (three times per week). Likewise, important results of greater than 70% reduction in lesion closure and amputation risk (which is less than 15% in groups 1 and 3) were obtained; i.e. the injury is mostly transferred from an amputated condition to a non-amputated condition.

Example 2 determination of rhEGF levels in plasma of DFU patients treated by the intralesional route

Pharmacokinetic studies were performed starting with blood draws on 31 patients. The design was aimed at determining kinetic parameters after application of rhEGF within the lesion in DFU. The most important parameters evaluated are, inter alia, the area under the curve (AUC) and the half-life (t)_1/2). The patient was treated three times a week with a drug containing rhEGF at the following levels: 25 μ g/dose, 75 μ g/dose and 225 μ g/dose. The treatment is carried out for 8 weeks or less if the area of the ulcer reaches 1cm or less²The size of (2).

The demographic characteristics of the patients are seen in table 3. 56.2% of patients are male. The average age was 63 years. 81.2% of patients are type 2 diabetes, with a wide range of variability in the time of progression of diabetes, ranging between 8 and 41 years.

TABLE 3 demographic characteristics of the patients participating in the study

^aSD: standard deviation;^bQR: four-bit pitch

Measurement of protein (rhEGF) as an active ingredient of the drug immediately before the first application (t)₀) Immediately after the first application (t)₁) And at different times after the first administration. By commercial ELISA kits (R&D) To do so by<The rhEGF concentration was determined in a plasma sample of the patient with a sensitivity of 0.7 pg/ml. For the determination of the growth factor, the instructions of the kit manufacturer were followed.

Two hour sample collection was sufficient to characterize the pharmacokinetic profile. Once this time is over, the value of rhEGF returns to the basal level in a manner independent of the dose received, without a subsequent increase.

It can be observed in table 4 that higher AUC values were detected at doses of 75 μ g and 225 μ g, compared to the values reached with the 25 μ g dose. Patients receiving this lower dose had lower t relative to the doses of 75 μ g and 225 μ g_1/2The value is obtained.

TABLE 4 pharmacokinetic parameters evaluated for different rhEGF doses administered to DFU patients

From the bioavailability analysis performed, it was observed that a small fraction of protein entered the blood when doses of 25 μ g and 75 μ g were applied. Approximately 19 μ g and 58 μ g, respectively, remained in the ulcerated area. On the other hand, for a dose of 225 μ g, the maximum amount of rhEGF immediately entered the blood, and only a small fraction stayed in the ulcer region. The fraction was about 63. mu.g. That is, an increase in the dose of the growth factor administered does not result in a greater concentration in the tissue. These results are consistent with the fact that an increase in EGF dose did not result in a greater effect.

Example 3 healing of DFU following administration of rhEGF intralesionally at various doses and application regimens

By considering the results obtained in examples 1 and 2, a study in DFU patients with criteria for diseased limb amputation was devised in which different rhEGF doses and administration regimens were explored, including the frequency of weekly administration of rhEGF once the patient no longer had criteria for amputation of the limb.

In one aspect, 48 patients were treated and three times weekly treatments with rhEGF were applied until no further criteria for diseased limb amputation were confirmed by evaluation. This period of repeated administration over one week is typically concurrent with the first three weeks of therapy with injectable rhEGF. Treatment with the growth factor is then continued once a week. The route of administration is always an intralesional route. Complete treatment is carried out for 8 weeks, or less, if the bed of ulcers is already covered by 100% of the available granulation tissue before these 8 weeks, or if the lesions have reached ≦ 1cm²The area of (a). The dose administered was 2.5. mu.g, 25. mu.g, 50. mu.g and 75. mu.g per group. 7 patients were included in the group receiving 2.5 μ g; 10 patients were included in the group receiving a dose of 25 μ g; 13 patients were included in the group receiving 50 μ g; and 18 patients were included in the 75 μ g group.

On the other hand, there was a group of 93 patients who were treated with a dose of 75 μ g three times a week throughout the period. The treatment period was 8 weeks. At the end of the study, the results of this patient group were used as a control, compared to the remaining groups that received the treatment regimen described in the previous paragraph.

Among patients, males predominate. The mean age was 64 years, and the predominant type of diabetes was type 2. The size of the ulcer is 22-27 cm²And in most patients the lesions are classified into the wagner classification at levels 3 and 4. Patients in all groups continued to receive conventional therapy, which was performed according to the treatment standards for DFU established by cuba national vasculology group, as outlined in example 1.

The results obtained are shown in table 5. It is important to emphasize that in those patients receiving the same rhEGF amount/dose, similar granulation results were obtained with the proposed new treatment regimen, compared to the prescribed application regimen three times a week over 8 cycles.

TABLE 5 results of intralesional application of rhEGF in patients treated with different dosages and application regimens

Objective granulation response: at 3 weeks, > 75% of the lesion area was covered by useful granulation tissue. Complete granulation response: at 8 weeks, 100% of the lesion area was covered by useful granulation tissue.

During follow-up, lesion closure was obtained in 4 patients (57%) in the 2.5 μ g group, 5 patients (50%) in the 25 μ g group, 9 patients (69%) in the 50 μ g group, and 13 patients (72%) in the 75 μ g group in the group receiving the three weekly and subsequently weekly combined regimen. In the treatment group acting as a control, in which patients received three times weekly applications of rhEGF (75 μ g), lesion closure was obtained in 81% of the individuals. Similarly, amputation was less than 12% in the four groups differing in rhEGF dose and in the group acting as a control. Surprisingly, the new treatment regimen suggested here, together with conventional therapy, results in a reduction of the risk of amputation to a degree of more than 80%.

This combination treatment regimen, while effective in patients, also allows for a reduction in the likelihood that these patients suffer from the most significant adverse events of intralesional administration of rhEGF (e.g., pain and burning, shivering, and chills), which is achieved by exposing the patients to fewer exposures to rhEGF administration via this route.

Example 4 healing of DFU following administration of rhEGF within weekly lesions

A series of cases to which conventional therapy was applied was treated, as described in example 1, with rhEGF at a dose of 2.5. mu.g (10 patients), 25. mu.g (15 patients), 50. mu.g (15 patients) or 75. mu.g (20 patients). The in-lesion therapy with the growth factor is performed once a week for 8 weeks or less if lesion closure is generated to 1cm or less²The size of (c). Another group of patients received only conventional therapy except that the activity was performed three times per week until complete closure of the lesion was reached.

Patients in this series of cases did not meet the DFU diseased limb amputation criteria at the time of initial evaluation. Typically, a larger percentage of patients for all groups are females, whether in those patients prescribed rhEGF or those patients for whom the drug is unspecified. The predominant type of diabetes is type 2, with a median evolution of 15 years. The size of ulcer is 2cm²And greater than 20cm²To change between. As reviewed, conventional therapy in all patients was performed according to the treatment standard for DFU established by the cuba national vasculology group.

Results for lesion closure between 8 and 12 weeks after initiation of treatment are shown in table 6. When comparing the treatment procedure comprising once weekly applied intralesional rhEGF with the conventional therapy only, surprisingly superior results were observed, which supported the administration of the growth factor in the four groups of patients. The lesion closure was 60% to 80% in all groups treated with rhEGF, whereas lesion closure was observed in 45% of individuals in the group receiving only conventional therapy.

TABLE 6 The percentage of lesion closure after intralesional administration of rhEGF in combination with conventional therapy or conventional therapy alone

Example 5 Effect of injectable rhEGF administration once weekly in DFU patients diagnosed without diseased lower limb amputation

To confirm the effect of once weekly intralesional administration of injectable rhEGF on DFU healing, patients who did not have the criteria for diseased lower limb amputation at the time of initial evaluation or diagnosis were treated.

Patient A: a 62 year old male diabetic patient with 15 years of diabetes evolution who exhibits a DFU with the following characteristics: superficial lesions that do not reach the deep level of the foot and have no signs of infection, at the time of physical examination and by hemodynamic parameters and transcutaneous oxygen pressure (TcPO)₂) Not of moderate or criticalIschemia is caused. The size of the injury after treatment is 10cm². rhEGF was administered to the patient for two weeks at a dose of 75 μ g, on a weekly frequency, and complete closure of the lesion was observed after the time elapsed.

Patient B: a male diabetic patient 54 years with 10 years of diabetes evolution, who exhibits a DFU with the following characteristics: deep lesions affecting the joint capsule and ligaments, but no infection nor associated osteomyelitis, at physical examination and through hemodynamic parameters and TcPO₂Without moderate or critical ischemia. The bed of ulcers is covered by more than 75% of the available granulation tissue. The size of the ulcer is 7cm². rhEGF was applied at a dose of 75 μ g once a week for two weeks. Complete granulation response was obtained at the end of this time, and lesion closure was obtained at 9 weeks after the first application.

Patient C: a 68 year old female diabetic patient with an 18 year progression of diabetes, exhibiting a DFU with the following characteristics: deep lesions affecting the joint capsule and ligaments, but without infection and associated osteomyelitis, at physical examination and through hemodynamic parameters and TcPO₂Without moderate or critical ischemia. The size of the ulcer is 7cm². rhEGF was not administered to the patient, but was treated with conventional therapy. Follow-up was maintained for the patients. In the absence of treatment with the drug, a complete granulation response was obtained at 14 weeks, and closure of DFU was not obtained at 20 weeks.